* Will Smith - Robert Neville
* Alice Braga - Anna
* Emma Thompson - Dr. Alice Krippin (uncredited)
* Charlie Tahan - Ethan
* Salli Richardson - Zoe Neville
* Willow Smith - Marley Neville
* Darrell Foster - Mike
* Abbey and Kona - Samantha (Sam), the dog
* Dash Mihok - Infected Alpha Male
* Joanna Numata - Infected Alpha Female
* Pat Fraley - President of the United States (voice)
* Mike Patton - Creature vocal effects
The film is set in the years 2009 and 2012. It opens in the fall of 2009 with a television news broadcast featuring Doctor Alice Krippin (Emma Thompson), who claims that she has created a cure for cancer with a 100% success rate. Dr. Krippin says that the cure was created by altering the measles virus.
In September 2012, U.S. Army virologist, Lieutenant Colonel Robert Neville (Will Smith) is left as the last healthy human in New York City and possibly the entire world. A series of flashbacks and recorded news broadcasts reveal that the genetically re-engineered measles virus (referred to as K.V. or Krippin virus) mutated into a lethal airborne strain that spread worldwide and killed 5.4 billion people, i.e. 90% of humanity. Of the 600 million survivors, only 12 million people possessed a natural immunity to the virus. The rest degenerated into bald, pale, aggressive beings referred to as "Darkseekers" (the DVD subtitles refer to them as hemocytes – actually a cell type found in an insect's immune system), who have a painful intolerance to UV radiation. This forces them to hide in buildings and dark places during the day. The "Darkseekers" exhibit increased speed, agility, aggression, and strength, as well as higher pulse, metabolism and ventilation rates, and increased body temperature. Despite their primal behavior, the Darkseekers seem to retain some basic problem-solving intelligence, animalistic cunning and the capacity to organize themselves. The remaining immune human population were killed by the infected. Flashbacks reveal that his wife Zoe (Salli Richardson) and daughter Marley (Willow Smith) died in a helicopter accident during the chaotic evacuation of Manhattan, prior to the military-enforced quarantine of the island in December 2009.
Neville's daily routine includes experimentation on captive infected rats to find a cure for the virus, and trips through an empty Manhattan to collect supplies from abandoned homes and hunting deer that 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 noon each day at the South Street Seaport. Neville's isolation is broken only by the companionship of his German Shepherd dog Samantha ("Sam"), interaction with mannequins he has set up as patrons of a video store, and recordings of old television broadcasts. Neville drives a Ford Mustang Shelby GT500, using it to aid him in hunting deer in the streets of Manhattan.
When one of his experiments on rats shows a promising treatment, Neville sets a snare trap and captures an infected woman. Back in his laboratory, located in the basement of his heavily fortified Washington Square Park home, Neville attempts to treat the infected woman, without success. Later, after finding one of his mannequins in front of Grand Central Terminal, he is himself caught in a trap and passes out. When Neville finally regains consciousness and manages to get free, it is dusk, and he is attacked by a pack of infected dogs. Although Neville and Sam manage to kill the dogs, one of the infected dogs bites Sam (although dogs are unaffected by the airborne strain of the virus, they are still affected by the contact strain). Neville takes the wounded Sam back to his lab and attempts to cure her, but she begins to exhibit symptoms, and he euthanizes her.
Later that night, after burying Sam in a cornfield, and overcome by grief and rage, he attacks a group of the infected. Despite killing a large number of Darkseekers, the infected overwhelm Neville and nearly kill him, but he is rescued by a pair of immune survivors, Anna (Alice Braga) and a young boy named Ethan (Charlie Tahan), who have heard his AM broadcasts. They 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. When Anna claims that their meeting wasn't coincidence, but part of God's plan, Neville argues that given the devastation incurred by the Krippin virus, there can be no God.