The Joker is the direct cause of the events of Injustice, and thus plays a major role in the first issues of the prequel comic, set five years before the events of the game. Prior to this, the Joker kills his fellow psychopath and Batman rogue Jonathan Crane (aka Scarecrow), stealing his signature fear toxin and dumping his body in S.T.A.R. Labs. At the Labs, he murders several scientists who had been experimenting on kryptonite and steals their samples in preparation for a grand scheme.
The Joker is first seen luring Lois Lane and Jimmy Olsen, Superman's wife and best pal respectively, to the Metropolis docks with a false tip-off of a juicy story. While the two are distracted by a disguised Harley Quinn, Joker kills Jimmy by shooting him through his camera lens. He happily greets a terrified Lois, as he and Harley ditch their disguises. He later hijacks a nuclear submarine that he takes far out into open sea with his lackey and their hostage. When the superhuman community learns of the Joker's location, Superman is first on the scene, scuttling the sub and tearing the hull open.
The Joker and Harley have just finished performing some kind of operation on Lois as the Man of Steel bursts into the sub. Before Superman can do anything, the Joker gasses him with a kryptonite-laced version of Scarecrow's fear toxin. As Superman tries to regain his wits, he suddenly sees Doomsday before him and tackles the monster into the sky. Joker tells Harley it's time to go, though his childish accomplice is reluctant to part with their stolen sub, that she named. He warns her that the do-gooders will arrive any moment, so she bids goodbye to 'Gunther' and the pair turns to escape.
Suddenly, Joker and Harley are pinned down by a giant hand projected from Green Lantern's ring. Green Lantern and Wonder Woman touch down just as Batman arrives, carried by the Flash. The Joker is overjoyed to see his nemesis and takes delight in mocking him for having been raced over on the Flash. When Batman investigates the sub and finds nothing, he demands to know Lois' location. Joker reveals that he performed an operation on Lois to turn her into a "trigger" - more specifically, the trigger to the submarine's nuclear warhead, which is now in the center of Metropolis and ready to detonate the moment Lois' heartbeat stops.
With none of the heroes able to warn Superman in time to avert the Joker's plan, the Clown Prince of Crime offhandedly comments that he discovered that Lois was pregnant while operating on her. This remark gets him punched in the face by Batman, but the plan goes off without a hitch; Superman unknowingly takes Lois into space, ironically coming to his senses just in time to watch his wife and unborn child suffocate in the vacuum. The Joker and Harley watch in glee as Metropolis is destroyed in a blast of nuclear fire.
In the wake of the horror, the Joker is locked up in Gotham Prison, where a furious Batman demands to know why his nemesis went so far as to take everything from Superman. The Joker comments that he didn't need a reason to do it, but nonetheless provides an explanation: he was never able to beat Batman, so he decided to play his games on an "easier" level. He taunts Batman further by claiming that destroying Metropolis was "as easy as killing a puppy with a kitten."
Suddenly taking a philosophical turn, the Joker asks Batman, "What do you think Superman will become?" The Caped Crusader firmly believes Superman will rise from the pain the Joker has inflicted on him - that the Man of Steel is incorruptible - but the Joker laughingly asks, "You honestly think your friend will still be fun to play with?" When Batman insists that Superman will recover, the Joker replies grimly, "No. I think it's going to be a whole lot funnier than that."
Moments later, Superman himself breaks through the wall, shoving Batman aside and grabbing the Joker. Mercilessly taunting Superman about the loss of everything he ever loved, the Clown Prince of Crime's portent comes to pass as he finally drives Superman past his breaking point. The Joker exhales a final maniacal laugh as Superman impales him through the chest with his entire arm. The colorful criminal dies with a smile on his face while Batman looks on in horror.