Misuzu in Air, in one of the most heart-wrenching anime scenes ever devised (the word "goal" will never hold the same meaning any more to anyone who has seen it). The fact that she might be reincarnated or freed from her curse does not diminish the fact that Misuzu herself is gone for good and leaves behind her immensely grieving adoptive mother, Haruko.
Black Cat Detective is almost schizophrenic about this. On the one hand, this show has no problem killing characters for real. The evil mice certainly die when they get killed. His second in command, well, if you watch this show, don't get to attached to him. But the elephant who gets shot in the middle of the head at point blank range is revived by an enormous syringe.
Played straight and subverted in Code Geass. One of the elements contributing to its Broken Base status was the fact that sometimes a character would appear to be Killed Off for Real, explicitly or at least implicitly, when in fact it was a case of Only Mostly Dead (Jeremiah, Mao, Guilford, and Cornelia) or Never Found the Body (Nunnally, Suzaku). The characters who DO die for real include, among others, Clovis, Mao (the second time), Euphemia, Shirley, Rolo, Emperor Charles, Marianne, Diethard and, finally (possibly), Lelouch.
Word of God says Lelouch has indeed been killed off for real.
Leomon from Digimonseason one and three. It's become a joke in the Digimon community that anything even remotely resembling a Leomon WILL die before series end, guaranteed. Also Oikawa, Black Wargreymon (could not be reborn due to being artificial), Arukenimon, and Mummymon from season two, as well as some family members of various characters that died before the seasons' start.
We might not see him again, but Leomon (Season 1 only) was presumably reborn, as Whamon was. Digimon who are destroyed get reborn in Primary Village. Except the evil Digimon, because they never come back to cause trouble after they are destroyed, so this trope applies to most of the evil Digimon as soon as they disintegrate.
There are three exceptions to this at first: Etemon seemingly died at the end of his arc, but returns as Metal Etemon in the Dark Masters' arc; however, two episodes into his return he is definitely destroyed by Saber Leomon (though he is the one who inflicts the mortal blow on him as well). Also, Devimon and Myotismon, while definitely killed in Season 1, returned as ghosts in Season 2 (Myotismon was even revived for the second time), but both were permanently destroyed in spirit form.
Wizardmon's death was, in fact, permanent. Because he died in the 'real world', there was no way for his data to be reborn. He did return as a ghost in Season 2, and disappeared after helping the Digidestined out.
Pumpkinmon and Gotsumon also got Killed Off for Real. That "dungeon" crap was just Dub Text; they clearly got killed in the real world just like Wizardmon courtesy of Myotismon's Bad Boss tendencies. (To fit this into the dub, one could say Myotismon changed his mind at the last second and killed them, which was even stated by Matt in the next episode)
Digimon Tamers counts as a meta example; all deaths are final as opposed to every other series where everyone can be reborn. It just so happened that Leomon was the first time a main characters Digimon got killed off.
The only exception is Vajramon who comes back for no reason the first time but is destroyed for good the second time.
In Digimon Frontier, though most destroyed Digimon are reborn as Digieggs (and purified in the case of the evil ones), the Big Bad, Lucemon, is destroyed for good the second time when his larval form is slashed by the spirits of the ten Legendary Warriors. Also, Golemon, Mercurymon's minions, Oryxmon, Sorcerymon, and apparently Crusadermon and Dynasmon are not reborn as Digieggs on death.
Dragon Ball, the franchise that consistently proves that Death Is Cheap, has succeeded in killing off several minor characters. Not focusing on the swaths of people that were killed that couldn't possibly have been brought back by the Dragon Balls, you also have several of the Kais. They were absorbed by Majin Buu, but due to being within him for millions of years, there's no way to remove them from his body. So when Buu dies, they officially die as well.
In the original series, this is justified with Grandfather Gohan, since he prefers to stay dead.
Frieza, Cell, and Buu, the main antagonists of Dragonball Z, are notable for surviving several attacks that seemingly kill them. Frieza survives Goku's Spirit Bomb, and later survives an energy blast from Goku that he and King Kai think killed him, and then Namek's explosion afterward. He returns as a cyborg, but is destroyed for good by Trunks. Cell can regenerate lost limbs, most notable instances being when Goku destroys his top half and when he selfdestructs, but in the end Gohan destroys him. As if Frieza being reduced to dust and Cell's energy vanishing completely wasn't enough, both are later shown in HFIL (Hell) with halos over their heads, further proving that they are dead. And no matter how often Buu is blown to bits, he always comes back together, but is finally destroyed by Goku with a Spirit Bomb.
Every un-reformed villain that dies stays dead, with the exception of Frieza, who reappears in  and Dragon Ball Super.
Guru is the only good guy who's killed and never comes back to life.
In GT, Piccolo insists on staying dead so that the Black Star Dragon Balls can never be used again. He continues to make several afterlife appearances, but it's stated explicitly that the Dragon Balls will never be used to bring him back to life again.
Fate/stay night has the protagonist, Shirou (potentially) killed off in the finale of Heaven's Feel. The Tear Jerker ending sticks to and expands on this, showing the epilogue through the eyes of Sakura as she ages and watches everything change around her, remaining alone until the end... The most depressing part of the game, hands down.
The entire Gundam franchise is absolutely notorious for this. With only a few exceptions, all of the series pull no punches when displaying the brutality of war. This is the series that gave rise to Kill 'em All after all.
The Universal Century timeline has this all over the place. Besides the millions upon millions who die in the background, major characters fall in some fantastically brutal ways. The most memorable of these comes during Mobile Suit Gundam: Char's Counterattack, where Amuro Ray and Char Aznable die at each other's hand in the climax.
The Cosmic Era is hands down the bloodiest and darkest of the Non-UC timelines. Gundam SEED in particular saw the death of more main characters than the most violent of the Universal Century series, Victory Gundam. Thanks to higher technological capabilities at the time of production, all of the carnage was visible in high definition.
Just when they're getting developed, Gundam 00 also has the deaths of both Lichty and Christina via point-blank gunshot from a mobile suit. It's made all the more tragic considering Lichty used his body to shield Christina, but she got a large piece of debris jabbed into her back anyway. Just in case there was any thoughts they could survive, the cockpit also exploded. This series is obviously fond of deaths by explosions.
And then there death of Ali Al-Sachez near the end of the second season. The original Lockon's brother, Lyle, tried to spare him but when Ali tried to use the opportunity to kill him, Lyle quickdrew his pistol and shot him in the head then casually shot him a few more times in the torso to make sure he was finished.
And let's not forget Nena. She got her antique mobile suit brutally dismembered into just the cockpit by Louise, who then stabbed the cockpit for good measure. Naturally, the last we see of Nena is of her cursing the hell out of her opponent with Blood from the Mouth before her cockpit explodes. Conversely, her brothers suffered brutal fates as well, with Michael suffering a One-Hit Kill and Johann lamenting as his unit is being destroyed.
InuYasha: There are several methods for returning the dead back to life and several characters that die end up bring brought back to life (a couple of characters even die twice and are restored to life twice). When Kikyou and Kagura end up dying in Redemption Equals Death storylines, the in-universe methods that exist can't save them as both of them end up being a case of No Body Left Behind. Subverted with Kohaku. He is set up to be this trope when it's confirmed the known methods for restoring the dead to life won't work on him. However, when it finally happens, a new one-off method turns up to restore his life, after all.
The mangaka of JoJo's Bizarre Adventure is not afraid to kill off his characters. Supporting protagonists usually have a good chance of dying; William Zeppeli from Part 1, Caesar Zeppeli from Part 2, and several of the primary protagonists' True Companions from Parts 3 and 5. Mostly averted in Part 4, which takes place in relatively tame circumstances compared to the other installments.
Even main characters can get this treatment when you least expect it. Jonathon Joestar sacrifices himself at the end of Part 1, Jolyne Kujo (along with everyone else on the good guys' side save for their Tagalong Kid) are killed just before the conclusion of Part 6, and Gyro Zeppeli (who shares main character status with Johnny Joestar) dies in Part 7.
Kekkaishi's Gen, much to the surprise of... everyone.
Another character who gets this treatment is Silcardo Junazard, the "Demon Fist" of the One Shadow Nine Fists. In this case it's a badly-needed case of Kill Him Already!, he violates the unofficial rules which stipulate that master-class fighters must not interfere in fights between two other masters and (more seriously) must not meddle in fights between two disciples, and crosses the Moral Event Horizon by manipulating Akira Hongo into almost killing Kenichi—almost because Hongo, who was fighting unconsciously at that time, realized what was about to happen and pulled back on his attack at the very last second—all so he can take advantage of Miu's resulting sense of mental chaos when she thinks Kenichi's been killed, kidnap her and brainwash her to become his newest student. In fact, the character is so bad in this regard that he ends up getting an utterly satisfying Karmic Death at the hands of a vengeful Hongo during an Enemy Mine with Ryozanpaku, possibly with sanction from the One Shadow (Miu's father) himself.
In the Super Dimension Fortress Macross/Robotech episode, "Pineapple Salad/Goodbye Big Brother," it should have been obvious what was going to happen with Hikaru Ichijou/Rick Hunter's senpai/brother, Roy Focker/Fokker, but the usual standard bowdlerization of 1980s American television animation made Western viewers assume that the show would take the safe route. When the show actually followed through and killed him off, it showed this was no ordinary animated series on North American TV. Moral Guardians raised a fuss and complained that it was bad for children.
There was no such ambiguity in the original Japanese version. Three ragged holes in his back and everything. And the reactions of the rest of the main characters seals the fact that he wasn't coming back. In fact, just to give Akira Kamiya more stick time as Focker, they had to bring him back in a prequel.
After pulling off a couple fakeout deaths and at least one deliberate death joke, Macross Frontier may have gone overboard in making sure viewers understood that Michael Blanc was dead. He gets stabbed in the chest with an alien claw that's almost as big as he is, and is then sucked out into hard vacuum through a self-sealing hull breach. No real way of coming back from that one. Not to forget the "coughs up blood, makes a final 'I love you' speech, does the Really Dead Montage and has a memorial shot at the end with his glasses." You know, because it's the only way to be sure.
Martian Successor Nadesico killed off Akito's friend and enthusiastic mecha pilot Guy Daigoji early on, and teased his return several times. The only time he ever appeared again was in a hallucination, and it wasn't Akito's. The character that did come back was a minor backgrounder. Another example of the show's recurring theme, "life is not like Super Robot anime".
In the Mazinger franchise some characters on both sides died permanently:
While Naruto has several Disney Deaths, it also has several examples of this trope, including The Third Hokage, Asuma, Jiraiya, Itachi, Pain, and Danzo to name a few. Some characters like Asuma and Itachi are brought back to life to fight in the Fourth Great Shinobi War, but they are closer to zombies than how they were before they died.
Shikaku, Inoichi & Ao - as well as everyone within the HQ of the Allied Shinobi Forces when it was obliterated by the Ten-Tails.
Neji's death is certainly here as well. To further prove the death, his curse mark disappears, which only happens when a Hyuuga dies. So no subverting the Disney Death here.
There's a common rule in One Piece: If you die in a flashback, you're Killed Off for Real. If you don't die in a flashback, chances are, you might pull off a Disney Death. Prime examples of the former are Bellemere, Hiruluk, Tom, Kuina, Usopp's mother, Robin's friends and mother, and Brook's pirate crew. However, the latter rule saw itself subverted, with Portgas D. Ace having fallen straight into this, and Whitebeard following two chapters later.
Following these, the rule seems to have expired: In Punk Hazard, two of the antagonists, Monet and Vergo, were killed.
Sabo is an exception to the flashback rule as while he appeared to have died in his flashback, Chapter 731 reveals that he did survive and is now a Revolutionary.
Where do we EVEN begin with Pandora Hearts? First we have Break's entire family he served as a knight wiped out by chains, then the Flower Girl, Phillipe West's Dad, Human Alice, her soul is still alive, she just doesn't have a physical existence after Oz rejected her and sent her to the Abyss, Oswald (dismembered by Oz when he was B-rabbit,), Lacie, who was sacrificed to the Abyss and is Deader Than Dead, Arthur Barma, Isla Yura and his cult, Elliot Nightray and his entire family save for Gil and Vincent, Cheshire Cat (although he's alive still),Rytas and her master and Gruner, Reim he lived. His chain can fake death, but it still leaves the user with a bunch of injuries., Jack, who was killed by Glen 100 years ago He's alive, his soul is just shattered into bits after being forced to live through a "Groundhog Day" Loop of his body de-aging over and over., Revis., Oz is thought this way to people who don't know he got out of Abyss. Shot by Gil, but he lived. Damn, why do so many characters die in this series?
In XYZ43, Lysandre also ends up killed following the fight between the Megalith Zygarde and Squishy/Z2's combined 100% Forme. Probably.
In SM021, the Stoutland that was taking care of Litten. Unlike other examples, this is the entire focus of the episode.
Puella Magi Madoka Magica killed off Sayaka while the protagonist, Madoka, willingly erases her existence from both the physical world and the memories of (almost) everyone who knew her. Mami and Kyoko were seemingly Killed Off for Real only to be brought back in the final episode.
In Rainbow, Sakuragi dies about midway through the series. This is especially surprising since he was The Hero, Big Good and possibly the protagonist as well.
Simoun has the Heroic Sacrifice death of Mamina. And to add insult to injury, the Simile carrying her coffin is shot down. Other characters are much more ambiguous: We have Rimone and Dominura, who Time Travel, then take off to parts unknown; Amuria, for whom they Never Found the Body; Onasia, who, well, who knows; Yun, who now exists in the sort of limbo the previous did; and Neviril and Aer who Ascend to a Higher Plane of Existence. Angulas also manages to die as a suicide bomber, but still show up as a body in the cockpit of an enemy Simoun later. Great series, but very very strange.
The character Cosmo is killed in the last episode of Sonic X. By a shot fired by Tails, using a cannon for which Sonic and Shadow were the ammo, no less. Though this is completely wiped out in the dub.
The same is true for the revolutionary, Molly, in the episode Molly's Dream. 4Kids even went so far as to digitally edit out her gravemarker in the final scene.
Emerl is also killed by Cream after she fails to bring him back to normal at the end of the Sonic Battle arc, since Gemerl doesn't exist in Sonic X he ends up as this.
In Soul Eater manga there Mifune, Tezca, Arachne and, Joe.
Though one of these deaths was faked to get cover.
Not as of chapter 86...
Tezca is a bizarre case because he seems to be somehow immortal. After he dies, his soul is still alive, can still appear in mirrors, implies that he can just get a new body, and still counts himself as being one of the Death Scythes.
Shinigami in ch109. Kid doesn't realise this until he returns from the final battle with Asura, and Excalibur 'helpfully' explains why it's all Kid's fault.
Kamina's death in Tengen Toppa Gurren Lagann. And Kittan, Nia, Balinbou... And the Rest. Though those were near the Grand Finale, so their deaths weren't quite as shocking as Kamina's, who died in the eighth episode and was featured as a really important character.
Lagann-Hen averted this by keeping the rest of Team Dai-Gurren (save for Kittan) alive until the final battle.
In a subversion of Never Say "Die", in the English dub of Yu-Gi-Oh!, Cecelia Pegasus is killed by an illness (prompting Pegasus to begin Duelist Kingdom so that he can get his hands on Yugi's Millennium Puzzle and resurrect her), Gozaburo and the Big Five are all destroyed along with the virtual world they are trapped in as it is destroyed (Noah isn't listed here because he is speculated to have saved his mind on a backup file, whether or not he did, and therefore invokes Never Say "Die"), and also Yami Marik, the Great Leviathan, Yami Bakura, and Zorc are all destroyed near the end of their arcs (although how far these four can be subverted to NeverDayDie is debatable. Ahmet is also eaten by a monster in a flashback during Season 5.
In Yu-Gi-Oh!: Capsule Monsters, if a Capsule Monster is destroyed, it's forever lost. This happens to many enemy monsters as well as to most of the heroes' monsters by the end of the game.
Yu-Gi-Oh! GX: In the original version, Edo Phoenix's adoptive father DD perished in his final confrontation with Edo (although the dub version had him make a threat which made his fate ambiguous).
The Seven Stars Assassins/Shadow Riders Camula and Abidos, after their respective defeats (Titan gets Dragged Off to Hell instead and Amnael/Daitokuji becomes a Spirit Advisor).
Season 3 has Professor Viper being killed off by Yubel after outliving his usefulness. Additionally, when everyone who died in the alternate dimension over the course of the season is brought back at the end, Echo and Amon Garam were not.
Divine was truly killed in Yu-Gi-Oh! 5D's, devoured by Earthbound God Ccarayhua (and strangely, the dub did very little to sugarcoat this). Divine clearly did not deserve to be spared; as far as Asshole Victims went, he was one of the biggest.
Divine was actually not killed. Word of God has proven that he is in jail. His being "eaten" was akin to all the human souls that were absorbed by the Earthbound Gods/Immortals. Thus, they returned at the end of the arc. The real "killed off for real" characters in that arc are Rex and Rudger/Roman. After that, it's every person aside from Zone in the apocalyptic future.
Yu-Gi-Oh! ARC-V has most character "deaths" represented by the character being sealed into a card, making it ambiguous whether they're truly dead or stuck in a state of And I Must Scream. Sergey Volkov, however, did not get this treatment, instead being beaten to death off-screen by angry Commons.