- Will Smith as Dr. Robert Neville, a virologist and the sole remaining human inhabitant in New York City, searching for a cure to the virus
- Alice Braga as Anna, a religious survivor of the outbreak.
- Charlie Tahan as Ethan, a young survivor of the outbreak
- Abbey and Kona as Samantha ("Sam"), Robert's German Shepherd and partner. Sam was a gift from Marley shortly before her death.
- Dash Mihok as Alpha Male, the leader of the Darkseekers
- Emma Thompson as Dr. Alice Krippin, the creator of the Krippin Virus.
- Salli Richardson as Zoe Neville, Robert Neville's wife who died shortly after the outbreak
- Willow Smith as Marley Neville, Robert and Zoe's six year old daughter who died shortly after the outbreak.
- Joanna Numata as Alpha Female, the Alpha Male's partner who is captured and experimented on by Robert Neville.
- Darrell Foster as Mike, a soldier who accompanied Neville during the initial stages of the outbreak
- Pat Fraley as voice of the President of the United States
- Mike Patton as voices of the Darkseekers, violent albino creatures infected by the Krippin Virus who are vulnerable to sunlight.
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