Adaptation Dye-Job: Anime Dean has black hair. Fangirls have been known to argue over whether Live Action Dean's hair is brown or blond, but they can probably agree it's not black. For that matter, he has brown eyes instead of green.
Likewise, Jess's hair color went from blonde to a reddish-brown.
Mary Winchester now has brown hair, which makes Dean look more like their father and Sam look more like their mother, whereas in the live action show it's the other way around.
Lily now has short brown hair instead of long and blonde.
All Just a Dream: Jess's reaction to being attacked by a spirit trying to kill her.
Camp Gay: There's a deputy chief in one episode who fits the trope to a T. He lets Sam and Dean out of jail for the reason that they "are so yummy!" Of course, this is thrown into doubt later in the episode, when we discover that he had other motives for freeing them.
Composite Character: The Yellow-Eyed Demon, possibly, given that during his first appearance he is referred to explicitly and exclusively as "The Devil".
Everybody Lives: "What Lives in the Lake", where the brothers figure out the monster is actually a Kappa who is saving people's lives and convince the townspeople to let it be.
Eye Scream: In "3 Years Ago", Bloody Mary finds Sam's eyes sufficiently reflective to use as a portal. She doesn't damage them, though. He does bleed from them when she turns her power on him.
Max in episode 11, to his stepmother. Almost.
Ink-Suit Actor: To varying degrees. Dean and Sam were modeled on their live action counterparts, though with more emphasis on capturing the character than the actor. Everyone else, no.
Kick the Dog: Max Miller's father is even more of a bastard in this version by virtue of killing Max's dog.
Missed Him by That Much: In a flashback episode where Sam is still at college, Dean and their father work a case revolving around Jess, his girlfriend. Rather than disrupt his life again, they decide to just proceed with the case. This involves a lot of Sam barely missing seeing his father or his brother, getting to a ridiculous level when they are one aisle over in the library, not talking quietly, and he doesn't notice a single thing.
Mythology Gag: In the anime version of "What Is and What Should Never Be", a photo shows that Dean is a fireman. He wasn't one in the live action version of that episode, but mentioned wanting to be one in the live action "Devil's Trap".
The Other Darrin: Jensen Ackles only dubs Dean in the final two episodes of the series (Jared Padalecki is Sam throughout). The change is barely noticeable, though.
Our Vampires Are Different: They even have one difference from the vampires in the original show, namely that they can have kids who become vampiric around puberty.
Pragmatic Adaptation: Most episodes that are taken whole cloth from the live action show, such as "The Alter Ego" ("Skin") and "Moonlight" ("Heart"), have at least one change to them to help maintain suspense and the plot twists.
For the English DVD Release, the original version was used to replace the cover version, and all of the guitar solos are the DVDs menu music.Justified because it was released through the same channels as the main series (Warner Bros, etc), and they had the licence from it that they could use.
Red Oni, Blue Oni: As you can see from the page picture, the official website takes it literally. Their clothes (though not their skin and hair) also tend to show it off in the actual show.
Red Right Hand: Lily's "killing with a touch" power is in her right hand.
Rule of Cool: Due to the nature of adapting a live-action show into an anime, a lot more moments that simply physically can't be done on-screen, either for budget or safety reasons, are added in.
For example: Bobby rescuing the Winchesters with a helicopter(!) in "Devil's Trap".
The Stinger: One in each episode. Sometimes irrelevant (a funny moment or the scene where the townsfolk wave goodbye) and sometimes vital to the next episode (like the ends of "Devil's Trap" and "All Hell Breaks Loose, Part 1").
Tragic Monster:Madison of episode 10 "Moonlight,is very much this." She was turned into a werewolf; and without even realizing it murdered her own boss, killed her stalker ex-boyfriend and sadly, in the end, she had to be killed by the Winchester brother's to prevent her from harming anyone else.
Woobie, Destroyer of Worlds: Max Miller of episode 11 "Nightmare;" He spent his entire childhood being beaten by his father.This left Max introverted and broken; when he finds out he has telekinetic powers he takes vengeance on his father and seeks to do the same to his mother as well for sitting back and doing nothing to stop him.