At a press conference held by Bruce Wayne to declare his opposition to Arkham City, Wayne is arrested by TYGER mercenaries and is imprisoned in Arkham City. Hugo Strange discloses his knowledge of Wayne's dual identity as Batman before releasing him into the criminal populace. While Strange prepares to commence "Protocol 10", Wayne
obtains his equpipment via airdrop from Alfred Pennyworth, allowing Wayne to become Batman. As Batman, he first saves Catwoman from being executed by Two-Face, who hopes to gain respect by murdering her. After Joker attempts to assassinate Catwoman, Batman tracks him to his hideout in the Sionis Steelworks, believing Joker may know the truth behind Protocol 10.
Batman learns from Joker's doctor that the unstable properties of the Titan formula are mutating in Joker's blood, gradually killing him. Joker captures Batman and performs a blood transfusion on him, infecting him with the same fatal disease. Joker also reveals that Gotham hospitals have been poisoned with his infected blood. Desperate to save himself and innocent citizens, Batman seeks out Mr. Freeze, who had been developing a cure but has since been kidnapped by the Penguin.
Batman pursues Penguin to the Cyrus Pinkney National History Institute and defeats his forces, his imprisoned monster Solomon Grundy, and ultimately the Penguin himself, before liberating Mr. Freeze. Freeze tells Batman that he has already developed the cure, but its instability renders it useless. Batman deduces that the restorative properties within the blood of Ra's al Ghul can complete the cure. Batman tracks one of Ghul's assassins to his underground lair in Wonder City, leading Batman into a confrontation with Ra's and his daughter Talia, Batman's former lover. With Ra's al Ghul's blood, Freeze is able to develop an antidote, but it is stolen by Harley Quinn before Batman can use it.
Batman returns to the Joker and finds him fully healed. While Batman and Joker fight, Strange activates Protocol 10, revealed to be a scheme to wipe out the entire population of Arkham City, destroying the criminal element of Gotham. The TYGER troops begin mass executions, while Strange launches missile strikes from his base in Wonder Tower. A missile hits the steelworks, burying Batman under rubble. Before Joker can take advantage of the situation, Talia arrives and offers him immortality in exchange for sparing Batman's life. After escaping with the help of Catwoman, Batman is convinced to put an end to Protocol 10 before pursuing Talia and Joker.
Batman infiltrates Wonder Tower and disables Protocol 10. Ra's al Ghul is revealed to be the true mastermind behind Arkham City, and mortally wounds Strange for failing to defeat Batman. With his dying breath, Strange activates "Protocol 11", the self-destruction of Wonder Tower. Batman and Ra's escape but Ra's commits suicide rather than risk capture. Joker contacts Batman, threatening to kill Talia unless Batman meets him at the Monarch Theater. Once Batman arrives, Joker demands the cure but is stabbed and apparently killed by Talia while distracted. While Talia is admitting to stealing the cure from Quinn, she is killed by a second Joker, who is still stricken with the disease. The healthy Joker reanimates into the shapeshifting Clayface, who has been masquerading as a healthy Joker at the ailing villain's request.
Batman incapacitates Clayface, but Joker blows up the theater floor, sending Batman plummeting into Wonder City below. Batman destroys Ra's' rejuvenating Lazarus Pit before the Joker can use it, and drinks a portion of the antidote. Batman debates curing his foe, but before he can act, Joker attacks him and inadvertently causes the antidote vial to smash. Batman says that despite everything Joker had done, he would have saved him. After Joker finally succumbs to his illness and dies, Batman carries his body out of Arkham City. As Commissioner Gordon questions the events, Batman places Joker's body on the hood of a police car and leaves in silence.
