- Lucas Till as Spider-Man
- Ellen Page as Gwen Stacy
- Matthew Modine as George Stacy
- Diane Keaton as Aunt May
- Richard Gere as Uncle Ben
- Richard Roxburgh as Kravin the Hunter
- Jason Statham as Scorpion
- Liam Hemsworth as Harry Osborn
- Kevin Costner as J. Jonah Jameson
- Ewan McGregor as Norman Osborn
- Phil Morris as "Robbie" Robertson
- Rob Lowe as Curt Connors
- Michael Massee as Otto Octavius
- Jake Abel as Flash Thompson
- Alex Daiddrio as Mary Jane Watson
Peter Parker graduates high school and gets a job working as an intern with his idols, Otto Octavius and Curt Connors. He gets bitten by a genetically-altered spider and falls unconscious. When he wakes up, he is in the hospital with Connors, Otto, Gwen Stacy (his girlfriend) and Aunt May. His Uncle Ben was driving to the hospital from work. On the way he sees a thug attacking someone, and Ben tries to stop him but is shot dead. Later, Pete discovers the powers he has gained and decides to use them to prevent things like Ben's death from happening again.
Meanwhile, in Africa, Sergei Kravinov has developed a serum that harnesses the altered DNA of an animal in order to give a human the traits and abilities of that animal. His entire shelf of chemicals falls on him, and he becomes Kravin the Hunter. He still judges the serum a success, and decides to go to America where he can find more test subjects. Meanwhile, the thief who killed Ben, who wants to not just be a petty thief, but rather a powerful enemy, is met by Kravin. When the thief expresses his desires to Kravin, Kravin injects him with the serum that gives him the powers of a scorpion. He also makes a suit with a mechanical acid-squirting tail for the thief to use. The thief decides to call himself "Scorpion." In return, he must work for Kravin.
Pete later confides his secret in Connors and together, they decide he should be called Spider-Man. The next day Peter sees a man rob a bank and stops him as Spider-Man. He gets pictures of them and sells them to J. Jonah Jameson at the Daily Bugle. Jameson pays him good money for them but doesn't hire Peter just yet. Spider-Man continues his adventures and Peter gets more pictures, and while many people love Spidey, others (including George Stacy) think he is bad. Meanwhile, Connors gets an invite for Peter to meet Connors' boss, Norman Osborn. Norman says he's heard great things about Peter, and hopes to one day hire him as an Oscorp employee. Peter sees Harry Osborn, Norman's son and his classmate. They kinda know each other, but after the interview they go and hang out and hit it off. They eventually become great friends, and (with May and Norman's approval) they move in together.
Meanwhile, Kravin discovers that the serum is unstable, and is causing his body to slowly become unstable. He sees Spider-Man on TV and decides to use Spider-Man's DNA to improve the serum.
The next day, Peter and May go to the police station where George Stacy tells them that they identified Ben's killer as Mac Gargan. George offers Peter and May his condolences and just as the two leave, Scorpion attacks the police station and holds everyone inside hostage, demanding that if Spider-Man doesn't show up within a half hour, he will kill everyone in the building. Peter, May, George, and Gwen (who was visiting her dad) are trapped in the building. Peter sneaks away and crawls n the roof until he reaches the restroom, where he changes into his Spider-Man suit. He swings out and attacks Scorpion. The two fight, and Scorpion sprays his tail, aiming for Spider-Man, but he accidentally hits the wall, burning a hole in it. Spider-Man tells the people to escape, and he distracts Scorpion. George tells May to take Gwen, and he shoots at Scorpion three times, but Scorpion's armor protects him. He hits George with his tail, knocking him into a wall and causing rubble to fall. Spider-Man webs up the rubble, and saves George, but Scorpion gets away. He reveals his identity to George, and George tells him to protect Gwen before he falls into a coma. Spider-Man takes him to a hospital, determined not to lose another father figure.