In one of the early story arcs in The Ultimates, Captain America temporarily drops the Incredible Hulk by landing a tank on him, beating him with his shield, then finishing with a solid kick to the groin that appears quite effective.
In a later issue, Cap's World War II sidekick Bucky comments on how funny he found it when he saw Cap 'kicking the Hulk in the stones'.
He did it again to nerd hulk in Ultimate Avengers.
In an issue of DC's Outsiders comic book, Checkmate operative Sasha Bordeaux attempts to break out of Nightwing's submission hold by going straight for His Most Sensitive Area. She's then very frustrated to find out that Nightwing wears an armor-reinforced athletic cup. Given that he's in a job where people try to beat him to death every single night of the week, wearing one only makes sense. It wouldn't be surprising to find out that such items are general issue for male superheroes now.
While in prison, Sasha also fended off another prisoner harassing her by kicking her in the crotch, leaving her doubled over.
One of DC's D-List characters, "Wild Dog," takes a shot to the crotch from a female reporter that drops him to the ground. One or two issues later, circumstances repeat, but Wild Dog informs the reporter that he now wears a metal cup. Apparently, none of Wild Dog's male criminal targets would even think of using this maneuver, so he never thought of incorporating that piece of protective gear before.
Batman and his associates seem so bizarrely fond of this that it becomes rather annoying - not just using it, but presenting it as some kind of unblockable, incapacitating technique that takes supposedly competent fighters who should be used to fighting dirty by surprise. A few examples:
Batman beat Karate Kid, the greatest martial artist in all time and space, skilled in dozens of styles some of which he invented, by kicking him in the groin. Batman commented that it was "cheating"...
Huntress punches Prometheus in the groin moments after he knocked out the globally feared fighter Lady Shiva, while he had the skills and reflexes of 30 of the greatest martial artists in the world at his disposal. Because apparently none of them know how to defend against a groin shot.
In Prometheus' first appearance Catwoman also hit him in the groin. With a bullwhip. She points out as she does so that the distinctive CRACK of a whip being used properly is a sonic boom caused by it exceeding the speed of sound.
At the same time, in Teen Titans "Titans East" (released summer 2007 in one volume) Nightwing and Robin (Dick Grayson and Tim Drake) stop by to rough up the Red Hood (Jason Todd), who came Back from the Dead during Infinite Crisis, because they believe he killed Duella Dent. Tired of their insinuations, Hood knocks Nightwing back, then kicks Robin and Nightwing both in the nuts. End of the scene has Robin kicking Red Hood in revenge before walking away.
This has been iconned as "The Gotham Handshake."
In an early issue of the Marv Wolfman/George Perez New Teen Titans, Robin (Dick Grayson) takes out a mind-controlled Cyborg by kicking him in the crotch. It was the early '80's in a code-approved comic, so while the art and dialog made it clear what is going on (Robin's thought balloon, for instance reads something like: "It's dirty fighting, but in this case...") they never actually show the kick, or describe it. They only show Cyborg's pained reaction; the kick also, inexplicably, knocks Cyborg out.
In Issue #4 of the 1986 Superman comic book limited series, The Man of Steel, Lois and Clark encounter a couple of South American terrorists onboard Lex's luxury liner during a party and even though the terrorists themselves are armed, Lois isn't afraid of them as she kicks one of them in the groin so that she can then get his gun and start to fight back against the terrorists until Superman arrives. The image of Lois' ballbust against the specific terrorist is used for the Badass Damsel trope page.
Also, on the cover of I'm your Man Part Two he is seen getting kicked in the groin by a very angry Steve Rogers, with a comically painful look in his face.
An issue of Spectacular Spider-Man had the Kangaroo dress in kangaroo-themed armor, complete with a laser cannon in the "pouch" — which happens to sit right at crotch level. Spidey webs the barrel. Cannon goes boom. (This was not the worst or most painful humiliation Kangaroo suffered in that story arc. In fact it was just a warm-up.)
It happened to Spidey too, once, courtesy of the female member of a rather lame team of villains called the Avant Guard, causing him to groan, "Not even Venom hit me there!"
A backup story had Syren using a "double kick" on a Mook— she high-jumps at them while delivering a kick to the head, and a knee to... the other head. Anatomically implausible unless you're fighting a dwarf, but it's a comic book, and she was fighting a leprechaun at the time.
In a team up issue with Wolverine and The Punisher, when Wolverine becomes too violent and is about to double cross him Punisher incapacitates him by blasting him in the groin with an Uzi, Wolverine holds it in pain and says, "Don't worry, they'll grow back".
Wolverine takes his revenge the same way on Sabretooth in Ultimate X-Men. Sabretooth is choking him with both hands and holding his head under water, when he suddenly doubles over in pain.
Wolverine: "At least I know to keep my family jewels covered in a fight."
In Doom Patrol #70, the main villain is a man known as The Codpiece, who attacks with a cannon attached to his groin. Read more about it here. And then played straight.
Luke Cage vs. Elektra in New Avengers. Elektra breaks her weapons against Luke's chest. Luke responds 'Matt says hello', and puts one big boot between her goalposts. Elektra crumples to the ground.
The tag "Inna Nertz" over at scans_daily has more than a few of these.
One of the people to take up the moniker of Stilt-man was killed when the Punisher shot him in the crotch with an RPGnote As in a Rocket-Propelled Grenade; he did not shoot a D&D Sourcebook at the villain's nethers, but that would've been pretty cool..
Supergirl kneed Powerboy in the crotch when they fought after she found out he was a creepy Stalker with a Crush. Since he also had superpowers, he didn't die...but probably wished he did.
What do you do, Mr. Baker, when you, as Animal Man, have your powers on the fritz and out of control, and Bloodrage, someone with some really funky lifedraining energy powers, has got you crushed and beaten on the floor? Go for the nutshot!
In Ultimate X-Men, Scott and Wolverine get into a huge fight over Jean. After knocking Wolverine unconscious on the floor, Scott is momentarily distracted by telling Kitty to get the Professor. A panel later, he screams and doubles over from a "low blow" attack from Wolverine, out of panel. And Wolverine says the only thing keeping him from using his claws in the attack was his love for Jean.
What happened when he saw Namora doing this to Atlas? He immediately proposed to her.
Sentry: Do you want me to unleash the power of a million exploding suns?
Hercules: No. I'll do it. (Herc kicks Sentry in a crouch)
Lampshaded and used against him in one arc. For plot reasons, Hercules and Thor are pretending to be each other, and Herc tells Thor he's not fighting enough like him. Thor's reply? "As you wish," right before he lets Herc have it in the nads.
Hercules: ::curled up in pain:: I have taught you well, my son.
In one issue of Sisterhood Of Steel, a male general donned a spiked codpiece and tried to get one of the Sisterhood's leaders to Groin Attack him. (She realized that something is wrong when a guy deliberately leaves his crotch open to attack, and kicked him in the jaw instead.)
Referred to (but not shown) in Fred Hembeck Destroys the Marvel Universe. The Guardian Angels formerly responsible for protecting the Marvel Universe's superhumans have been pulled off the job by God, who's mad because he saw a superhero ("Rumor has it it was Wolverine but I'm betting on She-Hulk") kicking a villain on the "wrong side of his jockey shorts."
Proving that he does not mess about, in PlanetaryElijah Snow gets attacked by Dracula and responds by freezing him solid...then proceeds to kick out his entire crotch. Ow.
In a Wolverine issue where he goes with Jubilee on a mission to find two criminals, eventually Wolverine tells Jubilee to kill them; she explains that she it's not her style and performs a double crotch kick on the criminals.
In Grant Morrison's The Invisibles, King Mob joyfully shoots an enemy guard in the crotch. The rest of the page features the guard's pain and horror as he realizes the impact this will have on his life.
Although Jesse Custer from Preacher tries his best to be a good guy, he has no qualms with going for the groin. In volume 7 a buff KKK thug threatens Jesse, describing how well he (the KKK thug) has bulked up with the help of lots of steroids; Jesse's response is to comment "Balls of mush" and squeeze his testicles so hard they bleed. Whether this falls under Crossing the Line Twice depends on personal taste. In the same storyline, there's an offscreen fight in which Jesse nutshots a man so hard his testicles recede permanently.
Also played more straight in the third volume, where one of the villains puts a round into Cassidy's groin as part of a torture. It wouldn't kill him outright, since Cassidy is a vampire with a potent Healing Factor, but it's the first in the many wounds the torturer inflicts that actually makes Cassidy cry in pain.
Sheriff Root, thanks to the Word of God had to go and fuck himself. Groin attack to the max.
And Herr Starr, who near the end has the entire package chewed off by a dog.
"My cock is on this bitch's mouth. And not in a good way."
Also Frankie, The Mafia hitman, who tells Starr about the time Russian mobsters cut his penis off with gardening shears (but left the testicles). His overly-macho family cut off all contact with him as a result. This manages to shock the normally amoral, self-absorbed Starr.
An issue of Ms. Tree had her confronted by an assassin wearing body armour who managed to disarm her and knock her to the floor. At this point, she realised he wasn't wearing a cup...
John Hartigan of the Sin City story, "That Yellow Bastard" deals with the scumbag rapist title character Junior as follows: "I take his weapons away from him...both of them." The first time he does this, he blasts Junior in the groin, but after his dad, the truly atrocious Senator Roark, pays to have it reconstructed (as well as sending Hartigan to prison on false charges of raping Nancy Callahan, the girl he saved from Junior), when Hartigan catches up to Junior again, he does this a second time by castrating him with his bare hands, Sonny Chiba style, before proceeding to beat Junior to death in a savage rage.
Marv also gets in on the groin-attacking action: "Here's a sign."