The story’s protagonist, Jake Sully (Sam Worthington), is a former Marine who was wounded and paralyzed from the waist down in combat on Earth. Jake is selected to participate in the Avatar program, which will enable him to walk. Jake travels to Pandora, a lush jungle-covered extraterrestrial moon filled with incredible life forms, some beautiful, many terrifying. Pandora is also home to the Na’vi, a sentient humanoid race that humans consider primitive, but are actually more evolutionarily advanced than humans. Standing three meters tall, with tails and sparkling blue skin, the Na’vi live in harmony with their unspoiled world. As humans encroach deeper into Pandora's forests in search of valuable minerals, the Na’vi unleash their formidable warrior abilities to defend their threatened existence.
Jake has unwittingly been recruited to become part of this encroachment. Since humans are unable to breathe the air on Pandora, they have created genetically-bred human-Na’vi hybrids known as Avatars. The Avatars are living, breathing bodies that are controlled by a human "driver" through a technology that links the driver's mind to their Avatar body. On Pandora, through his Avatar body, Jake can be whole once again. Sent deep into Pandora's jungles as a scout for the soldiers that will follow, Jake encounters many of Pandora's beauties and dangers. There he meets a young Na’vi female, Neytiri, whose beauty is only matched by her ferocity in battle.
Over time, Jake integrates himself into Neytiri's clan, and begins to fall in love with her. As a result, Jake finds himself caught between the military-industrial forces of Earth, and the Na’vi, forcing him to choose sides in an epic battle that will decide the fate of an entire world.
- Sam Worthington as Jake Sully. Cameron cast the Australian actor after searching the world for promising young actors, preferring relative unknowns to keep the budget down. Worthington auditioned twice early in development, and he has signed on for possible sequels. Cameron felt because Worthington had not done a major film, he was "game for anything", giving the character "a quality that is really real. He has that quality of being a guy you'd want to have a beer with, and he ultimately becomes a leader who transforms the world."
- Zoe Saldana as Neytiri, a female Na'vi Jake initially betrays, but eventually falls in love with. The character will be entirely computer generated. Saldaña has also signed on for sequels.
- Sigourney Weaver as Dr. Grace Augustine, a botanist who mentors Jake Sully. Weaver dyed her hair red for the part. Her character was named "Shipley" at one point. The character reminded Weaver of Cameron, being "very driven and very idealistic".
- Michelle Rodriguez as Trudy Chacon, a retired Marine pilot. Cameron had wanted to work with Rodriguez since seeing her in Girlfight.
- Giovanni Ribisi as Parker Selfridge, a passive-aggressive character.
- Joel David Moore as Norm Spellman, an anthropologist who studies plant and nature life (like Weaver's character).
- CCH Pounder as Moha, the Na'vi queen.
- Stephen Lang as Marine Corps Colonel Quaritch. Lang had unsuccessfully auditioned for a role in Cameron's Aliens (1986); the director remembered Lang and cast him in Avatar. Michael Biehn was considered for the role of Colonel Quaritch. He met with James Cameron three times and saw some of the 3D footage, but in the end it simply came down to the fact that Cameron didn't want people thinking it was Aliens all over again, as Sigourney Weaver had already been cast.
- Dileep Rao as Dr. Max Patel.
- Matt Gerald as Corporal Lyle Wainfleet, the main villain.