Embedded with fierce action and intense emotion, this fantasy superhero drama revolves around high-school senior Peter Parker visits a genetics laboratory with his friend Harry Osborn and love interest, Mary Jane Watson for a school field trip. There, Peter is bitten on the hand by a genetically engineered "super spider". Shortly after arriving home to his Aunt May and Uncle Ben, he goes unconscious.
Meanwhile, Harry's father, scientist Norman Osborn, owner of Oscorp, is attempting to preserve his company's critically important military contract. He experiments on himself with a new but unstable performance-enhancing chemical vapor that increases his speed, strength, and stamina. However, it also drives him insane and he kills his assistant, Mendel Stromm.
The next morning, Peter finds that his previously impaired vision has improved and his body has metamorphosized into a more muscular physique. At school, he finds that his body can produce webs and his quickened reflexes let him avoid injury during a confrontation with bully Flash Thompson. Peter discovers he has developed superhuman speed, strength, the ability to stick to surfaces, and a heightened ability to sense danger.
Brushing off Uncle Ben's advice that "With great power comes great responsibility," Peter enters a wrestling tournament to get money to buy a car and impress Mary Jane. He wins his match, but the promoter cheats him out of the contest money. When a thief steals money from the promoter, Peter accidentally allows the thief to escape. He later discovers that Uncle Ben has been carjacked and shot dead. Peter confronts the carjacker, only to realize it was the same thief he let get away. After Peter disarms him, the fleeing carjacker falls out a window and dies. Meanwhile, Norman Osborn kills several scientists and the military's General Slocum.
Upon graduating, Peter begins using his abilities to fight injustice, donning a costume and the persona of Spider-Man. Newspaper editor J. Jonah Jameson hires him as a freelance photographer, since Peter is the only person providing clear images of Spider-Man.
Norman, upon learning Oscorp's board members plan to sell the company, assassinates them at the World Unity Fair. Jameson quickly dubs the mysterious killer the Green Goblin. The Goblin offers Spider-Man a place at his side, but Spider-Man refuses. At the Osborn and Parkers' Thanksgiving dinner, Norman deduces Spider-Man's true identity after noticing a cut on Peter's arm which he saw on Spiderman; the Green Goblin subsequently attacks and hospitalizes Aunt May.
Mary Jane admits she has a crush on Spider-Man, who has rescued her on numerous occasions, and asks Peter whether Spider-Man ever asked about her. Harry, who loves Mary Jane, arrives and learns she does feel likewise towards Peter. Devastated, Harry tells his father that Peter loves Mary Jane, unintentionally revealing Spider-Man's biggest weakness.
The Goblin holds Mary Jane and a Roosevelt Island Tram car full of children hostage alongside the Queensboro Bridge. He forces Spider-Man to choose who he wants to save, and drops Mary Jane and the children. Spider-Man manages to save both Mary Jane and the tram car, while the Goblin is pelted by civilians showing loyalty to Spider-Man. The Goblin then grabs Spider-Man and throws him into an abandoned building where they fight. When the Goblin boasts about how he will later kill Mary Jane, an enraged Spider-Man overpowers the Goblin.
Norman regains control of his insanity and reveals himself to Spider-Man. He begs for forgiveness, but his Goblin persona attempts to remote-control his glider to impale Spider-Man from behind. The superhero dodges the attack, causing the glider to accidentally impale Norman instead. As he dies, Norman tells Peter not to tell Harry about the Green Goblin. Spider-Man takes Norman's body back to Norman's house and hides the Green Goblin's equipment.
At Norman’s funeral, Harry swears vengeance toward Spider-Man, believing he is responsible for killing his father, and asserts that Peter is all the family he has left. Mary Jane confesses to Peter that she is in love with him, but Peter, feeling that he must protect her from the unwanted attentions of Spider-Man's enemies, hides his true feelings and tells her that they can only be friends. As Peter leaves the funeral, he recalls Ben's words about responsibility, and accepts his new life as Spider-Man.