Socially awkward teenager Peter Parker grapples with his past and struggles with the mystery of his parents' disappearance when he was a child. His quest for answers leads him to his father's old partner, Dr. Curt Connors, at Oscorp Industries. While there, Peter is bitten by a genetically-altered spider, which endows him with extraordinary powers including superhuman strength, agility, and the ability to cling to most surfaces. He subsequently crafts a web-shooting device that allows him to swing between buildings and subdue foes. As he embraces his new identity and begins to understand the responsibility it carries, Peter constructs a persona to protect his identity and vows to use his powers for the good of others, becoming a vigilante hero in the process.

While adapting to his newfound abilities, Peter forms a bond with his high school crush, Gwen Stacy, whose father is the police captain tasked with apprehending the masked vigilante. Meanwhile, Dr. Connors, in his quest to regenerate his lost limb, experiments with a serum derived from reptilian DNA. The experiment goes awry, transforming him into a monstrous creature known as the Lizard. As the Lizard begins a campaign to turn others into similar creatures and chaos ensues in the city, Peter faces the dilemma of how to stop Connors without hurting him, while also preserving his secret identity and protecting those he cares about.