On a high school field trip, teenager 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 falls ill upon returning home. Meanwhile, at the chemical corporation Oscorp, Harry's father and company CEO Norman Osborn seeks to secure a military contract by testing a performance-enhancing chemical on himself. The chemical causes him to go insane and kill one of his scientists.
The next day, Peter develops spider-like abilities, including enhanced strength, senses, agility and speed, organic webbing in his wrists, and the ability to cling to walls. Hoping to buy a car to impress Mary Jane, Peter wins an underground wrestling event, but is cheated out of his earnings. Soon after, Peter's uncle Ben is killed by a man who robbed the wrestling event, and whom Peter let escape. Peter tracks down the killer, who eventually falls to his death. Meanwhile, a crazed Norman sabotages a product test by an Oscorp rival and kills several people.
Upon graduating, Peter begins using his abilities to fight crime. He creates a spandex suit and mask, and adopts the alias "Spider-Man". J. Jonah Jameson, the publisher of the Daily Bugle newspaper, hires Peter as a freelance photographer, since he can provide high-quality images of Spider-Man. When Oscorp's board of directors decides to oust Norman and sell the company, Norman assassinates them while wearing a disguise. Peter fends off Norman and rescues Mary Jane. Afterwards, Jameson bestows the name "Green Goblin" upon the mysterious masked killer.
Norman offers Peter a place at his side, but Peter refuses. They fight, and Peter flees after being wounded. Peter's aunt May invites Mary Jane, Harry, and Norman over for Thanksgiving dinner. Norman sees Peter's injury and realizes that Peter is Spider-Man. Later, Norman attacks and injures May, who is hospitalized. Peter is still unaware of the Goblin's identity, but realizes that the Goblin is targeting his loved ones. While visiting the hospital, Mary Jane confesses to Peter her infatuation with Spider-Man, who has rescued her twice. Harry, who is dating Mary Jane, sees her holding Peter's hand and assumes she has feelings for him. A distraught Harry tells his father that Peter loves Mary Jane.
Norman captures Mary Jane and a tram car full of children. He tells Peter to choose whom to rescue, then drops them both from a deadly height. Peter saves everyone, then lowers them to a nearby ferry for safety. An enraged Norman throws Peter into a building, then brutally beats him. After Norman reveals his intentions to kill Mary Jane, Peter finds the strength to fight back. Norman reveals his identity and begs for forgiveness, while discreetly preparing to impale Peter with his glider. Warned by his spider-sense, Peter dodges the attack, and the glider fatally skewers Norman instead. Before dying, Norman begs Peter not to reveal his identity to Harry. Peter takes Norman's body to the Osborn house, where he is confronted by Harry, who grabs a gun. Before Harry can fire, Peter escapes.
At Norman's funeral, Harry vows revenge on Spider-Man, whom he holds responsible for his father's death. Mary Jane then confesses to Peter that she loves him. Peter, however, feels he must protect her from his enemies, so he hides his true feelings and tells her that they can only be friends. As Peter leaves, he recalls his uncle's words: "With great power comes great responsibility."