On a school trip, high school senior Peter Parker visits a Columbia University genetics laboratory with his friend Harry Osborn and his love interest Mary Jane Watson. There, Peter is bitten by a genetically engineered spider, and he falls ill upon returning home. Meanwhile, Harry's father Norman Osborn, a scientist and the founder and owner of Oscorp, tries securing an important military contract. He tests a performance-enhancing chemical on himself and goes insane, killing his assistant.
The next day, Peter develops spider-like abilities, superhuman strength, sharper senses, agility, speed, and the ability to cling to walls. Ignoring his Uncle Ben's advice that "with great power comes great responsibility", Peter considers buying a car to impress Mary Jane. Despite winning an underground wrestling event to pay for the car, Peter is cheated out of his earnings. After Ben is killed moments later by a thief that Peter allowed to escape, Peter pursues him, but the thief falls out of a window to his death. Meanwhile, a crazed Norman interrupts a test of an exoskeleton created by Oscorp's rival Quest Aerospace and destroys the exoskeleton, killing several people.
Upon graduating, Peter begins using his abilities to fight crime, donning a spandex costume and the masked persona of Spider-Man. J. Jonah Jameson, publisher of the Daily Bugle newspaper, hires Peter as a freelance photographer, since he is the only person providing clear images of Spider-Man. Upon learning that Oscorp's board plans to oust him to sell the company to Quest, Norman assassinates them. Jameson names the mysterious killer the "Green Goblin."
The Goblin offers Peter a place at his side, but he refuses. They fight, and Peter is wounded. At Thanksgiving dinner, Peter's Aunt May invites Mary Jane, Harry, and Norman. During the dinner, Norman sees the injury and realizes Peter's identity. Believing the only way to defeat Peter is to attack those who are special to him, Norman later attacks and injures May. While visiting May at the local hospital, Mary Jane admits her infatuation with Spider-Man, who has rescued her on two occasions. Harry, who is dating Mary Jane, sees her holding Peter's hand and assumes she has feelings for him. Distraught, Harry tells his father that Peter loves Mary Jane, unknowingly revealing Spider-Man's biggest weakness.
Norman holds Mary Jane and a Roosevelt Island Tram car full of children hostage alongside the Queensboro Bridge. He forces Peter to choose whom he wants to save and drops them both. Peter saves both Mary Jane and the tram car and manages to lower them down to a nearby ferry for safety. An enraged Norman grabs Peter and throws him into the nearby Smallpox Hospital building and brutally beats him. When Norman brags about how he will kill Mary Jane, Peter gains the upper hand, and Norman reveals himself and begs for forgiveness while subtly getting his glider ready to impale Peter from behind. Warned by his spider-sense, Peter dodges the attack, and the glider mortally impales Norman instead. Norman begs Peter not to reveal his identity as the Goblin to Harry before dying. Peter takes Norman's body to the Osborn house and is confronted by Harry, who pulls a gun on him, but Peter escapes.
At Norman's funeral, Harry swears vengeance on Spider-Man, whom he falsely holds responsible for his father's death. Mary Jane confesses to Peter that she loves him. Peter, however, feels he must protect her from the attention of his enemies, so he hides his true feelings and tells Mary Jane that they can only be friends. As Peter leaves, he recalls Ben's words and accepts his new responsibility as Spider-Man.
