After the events of Resident Evil: Extinction, the Alice clones (Milla Jovovich) attempt to kill Albert Wesker (Shawn Roberts) at his base in Japan. All the clones are killed in an explosion as Wesker escapes in an aircraft. The original Alice ambushes him, but Wesker injects her with a serum that neutralizes the T-virus in her system, eliminating her superhuman powers and making her human again. Before he can kill her, the aircraft crashes and Alice emerges from the wreckage.
Six months later, Alice follows a repeating emergency broadcast from a survivors' safe haven known as "Arcadia" in Alaska. After many fruitless months searching for Arcadia and finding no survivors along the way, including her friends Claire Redfield (Ali Larter) and K-Mart (Spencer Locke), Alice begins to lose hope, believing herself to be the last surviving human on the planet. Stopping on an abandoned beach, Alice finds the helicopter taken by Claire and the others when they and Alice parted ways. A crazed Claire, under the influence of an Umbrella Corporation device attached to her chest, attacks her. Alice removes the device, finding that it has damaged Claire's memory, which she slowly regains.
Flying a two-person plane, they enter the ruins of Los Angeles and find a small group of survivors living in a maximum security prison, surrounded by the undead. Alice crash lands the plane on the roof of the prison and learn that Arcadia is not a fixed place but actually a cargo tanker traveling along the coast. Since the plane cannot take more than two, Alice and the survivors try to figure out a means to make it to Arcadia together. The survivors have been keeping watch on a prisoner, Chris (Wentworth Miller), whom they found locked in a maximum security cell when they arrived. He says that he is a soldier who fought the early outbreak, but was locked up by prisoners as a cruel joke when the mission failed and knows a way to reach the coast, and thereby Arcadia, but will not reveal it unless he is released.
They do not believe him, and keep him locked up until the undead manage to tunnel through the sewer ways and up into the prison; Alice and survivors are out of time to find a means to reach Arcadia so they decide to free him and use his escape route. He recognizes Claire and reveals himself as her brother, though she does not remember him. Chris' proposed method, a stored military vehicle, is legitimate, but the vehicle is not operational and they are left with no other option but to use the tunnel to escape through the sewers, and storm drains which empty at the coast. The group fights their way out — Alice, Claire, and Chris manage to escape into the sewers.
They continue to Arcadia, finding the ship completely functional, but abandoned. Exploring its inner depths, they realize that it is a trap set by Umbrella to lure survivors to the ship to conduct experiments on them. Claire now remembers what happened to her group when they arrived to Alaska: Umbrella ambushed their group when they landed on the beach, attaching the devices to them, though she managed to escape. They release the survivors, among them K-Mart, and Alice continues deeper into the ship, finding escape helicopters and a purging bomb. Deeper inward, she finds Wesker, who explains that he survived the crash thanks to the T-virus, which has also mutated him, granting him super human abilities. However, he finds it difficult to control. By assimilating Alice, the only individual to bond successfully with the T-virus, he will be able to gain full mastery of the T-virus.
Chris, Claire, and Alice battle the super-powered Wesker and seemingly kill him. Wesker does not die, however, but rather regenerates himself and makes his escape via a helicopter. He activates the purge bomb on the Arcadia in attempt to destroy the ship and kill the remaining survivors, only to find that Alice had hid the bomb on board his helicopter, which is promptly destroyed by the explosion. Alice, Claire, and Chris watch the destruction from the deck of the Arcadia, and momentarily blinded by the flash, they fail to notice a figure parachuting away from the helicopter.
Alice resolves to turn Arcadia into a real safe haven and broadcasts its message for any other survivors. As Claire, Chris, and Alice decide how to proceed with all the survivors, they see an approaching Umbrella assault fleet led by Jill Valentine (Sienna Guillory), who now is under the control of the same Umbrella device used on Claire