The film begins in the fall of 2009 on an interview talking about the cure rate from the "new" Krippen drug (named after the scientist (Emma Thompson) who created it). In September 2012, U.S. Army virologist Lieutenant Colonel Robert Neville (Will Smith) is left as the last healthy human in New York City. Three years earlier, a cure for cancer made from a re-engineered measles virus was developed with a 100% success rate. However, the virus mutated into a lethal strain that spread worldwide and killed 5.4 billion people (90% of humanity). Of the 600 million survivors, only 12 million people were naturally immune to the virus. The rest degenerated into bald, pale, aggressive beings referred to as "Darkseekers,"[4] who hunted down the immune humans as prey. The "Darkseekers" are so-called for hiding in buildings and dark places during the day due to a painful intolerance to UV radiation. The infected exhibit increased speed, agility, aggression, and strength. These abilities stem from an increased metabolic rate, which also consumes the infected with an overwhelming hunger which makes them resort to cannibalism. Despite their primal behavior, the Darkseekers seem to retain some basic problem-solving intelligence, animalistic cunning, and the capacity to organize themselves. Neville, who lost his wife Zoe (Salli Richardson) and daughter Marley (Willow Smith) in a helicopter accident during the chaotic quarantine of Manhattan in December 2009, has a daily routine that includes experimentation on infected rats to find a cure for the virus and trips through an empty, decaying Manhattan to collect supplies from abandoned homes and hunt deer that have moved into the city. He also waits each day for a response to his continuous recorded AM radio broadcasts, which instruct any uninfected survivors to meet him at mid-day at the South Street Seaport. Neville's isolation is broken only by the companionship of his female German Shepherd Samantha ("Sam") and interaction with mannequins he has set up as patrons of a video store.
When one of his experiments on rats shows a promising treatment, Neville sets a snare trap and captures an infected woman, as an enraged Darkseeker, the alpha male of the pack, locks eyes with him. Back in his laboratory, located in the basement of his heavily fortified Washington Square Park home, Neville attempts to cure the infected woman without success.
The next day, after finding one of his mannequins was apparently moved to the front of Grand Central Terminal, he is caught in a trap and passes out. When Neville regains consciousness and manages to get free, it is dusk and he is attacked by a pack of infected dogs set on him by the alpha male. Although Neville and Sam manage to kill the dogs, one of them bites Sam. The dogs are immune to the virus when it is airborne but not when there is blood contact. Initially Neville brings Sam home and injects her with a strain of his serum, but when she shows signs of infection and tries to attack him, Neville is forced to euthanize her by strangulation.
The next night, overcome by grief and rage after burying Sam, Neville attacks a group of the infected with his car, intending to end his misery by killing as many as he can. Despite killing a large number of Darkseekers, they overwhelm Neville and nearly kill him before he is rescued by a pair of immune survivors, an adult woman named Anna (Alice Braga) and a young boy named Ethan (Charlie Tahan), who have heard his radio broadcasts. Anna and Ethan take the injured Neville back to his home, where Anna explains that they are making their way to a putative survivors' camp in Bethel, Vermont.
The next night, the alpha male leads a large group of infected in an attack on the house, having followed Anna and Neville back the night before. The Darkseekers begin by charging at the house. Neville tries to use strong UV light to push them back, but the infected easily tear through the lights and continue to charge. Neville then uses planted mines. At first it works, but there are more Darkseekers than Neville thought, and they start getting closer to Neville's home. Neville tries to find Anna and Ethan. Before he can, he faces off one-on-one against the alpha male, nearly losing before forcing him to fall back. Neville realizes that this was just to distract him as another Darkseeker got into the house and tore at the roof from the inside so that the other infected can gain entry. After saving Anna and Ethan they hear the infected climbing the house, so they retreat into the basement laboratory. They seal themselves in a room with the infected woman, where they discover that Neville's treatment is working: the subject has reverted to a more human form. Unfortunately, the infected break in and the alpha male begins to break through the plexiglass separating them. Discovering that the last treatment has been successful, Neville draws a vial of the infected woman's blood and gives it to Anna before shutting Anna and Ethan in the coal chute. He uses an M67 hand grenade to wipe out the attackers at the cost of his own life. Anna and Ethan escape to Vermont and locate the survivors' colony, where Anna hands over the cure. In a voice-over, Anna claims that the survivors are Neville's legacy, as his fight for a cure became legend.
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