Alice attempts to kill Albert Wesker at his base in Tokyo with her army of clones. Wesker escapes and destroys the base, killing all of the clones. However, the real Alice boarded his aircraft beforehand. Wesker uses a serum to remove all of Alice's powers before the lack of a pilot makes the aircraft crash. Alice somehow survives the crash while Wesker seemingly perishes. Six months later, Alice travels back to North America in an airplane following emergency broadcasts from a safe haven known as ''Arcadia'' in Alaska (written of in the diary she found in the previous film). En route, Alice is attacked by Claire Redfield, who is being controlled by a device (placed on her by Umbrella); the device's destruction causes Claire to have amnesia. The two travel to the ruins of Los Angeles in a Yak-52 plane, where they find a group of survivors living in a prison surrounded by the infected. Luther West, leads the small band which includes Wendell, Crystal Waters, Bennett and Kim Yong, Angel Ortiz, and Chris Redfield, a prisoner under lockdown in the facility.

With their help, Alice lands her plane on the roof of the prison and learns that ''Arcadia'' is not a fixed place but actually a cargo tanker traveling along the coast. A disturbing piece of news is that ''Arcadia'' has stopped all broadcasts and has remained still. Alice and the survivors decide to find an alternate means to make it to the ''Arcadia''. Chris reveals he's Claire's brother, insisting he fell victim to a cruel joke by some escaped prisoners, and that he knows an alternate route. While in the shower, Alice is attacked by a group of Majini infected that managed to tunnel into the prison. Wendell, who was trying to peep on Alice, is taken away/eaten by the zombies. Alice, seeing as they are out of options, decides to set Chris free and leave the prison.

Chris reveals that the prison keeps an armored car that can be used to escape. However, a giant axe-wielding monster begins breaking down the gate outside. To overcome the problems, Alice, Chris and Crystal go deeper into the prison to retrieve additional guns from the armory; Luther and Claire attempt to reinforce the gate to keep the Axeman and other zombies at bay. As Alice, Chris and Crystal make it to the armory, zombies swarm them, overpowering and devouring Crystal. Angel informs Bennett and Yong the vehicle is missing its engine and it would take a week to fix. Panicking, Bennett shoots Angel, abandons Yong, and heads for ''Arcadia'' in Alice's airplane, slaying some zombies along the way due to poor piloting skills. Below, the Axeman breaks down the gate, allowing the zombies to enter the facility. With no other choice, the group decides to use the zombie-dug tunnels to escape into the sewers. Yong is suddenly cut in half by the Axeman, whom Alice and Claire then face. Alice attempts to fight it first and it knocks her out; Claire then defends Alice and just before it kills her, Alice awakes and saves them both. The two meet back up with Chris at the end of the sewers but they lose Luther to a zombie who drags him back into the sewers.

Alice and the Redfields commandeer a boat and board the ''Arcadia'', discovering it's functional but abandoned. Claire suddenly regains her memory, and reveals ''Arcadia'' was an Umbrella trap to gather survivors to be used as test subjects; they haven't learn from their mistakes. The trio begin releasing survivors, among them K-Mart. Seeing a trail of blood leading from an empty test subject container, Alice follows it deeper into the ship. As she expected, Alice finds Wesker, revived by the T-virus. However, Wesker is battling the virus for control of his body, something he believes could be pacified by DNA; this made the Umbrella staff flee in terror. As Alice is the only person to bond at a cellular level with the T-virus and successfully retain control, Wesker plans to eat her to assimilate this ability.

The Redfields arrive and attack Wesker while Alice battles two dogs and Bennett, who has allied himself with Wesker. Wesker easily overpowers Chris and Claire, but Alice is able to defeat both Bennett and Wesker with help from K-Mart. While leaving, they lock Bennett in the room with Wesker's corpse; Bennett is devoured by Wesker when he revives. Wesker then escapes in an aircraft, activating a bomb on the ''Arcadia''; the plane explodes instead as Alice placed the bomb there, thinking it might be set off.  Unbeknownst to them, a figure parachutes away from the explosion, while Luther emerges from the sewers, battered but alive. Alice resolves to turn ''Arcadia'' into a real safe haven and broadcasts a new message for any other survivors. Claire, Chris, K-Mart, and Alice soon see an approaching Umbrella assault helicopter squadron.

During the post-credits scene in one of the helicopters, Jill Valentine, who went missing after ''Resident Evil: Apocalypse'', is dictating the attack wearing the same mind control device used on Claire, which slightly glows at the end of the scene, thus setting up the events of ''Resident Evil: Retribution''.

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