Peter Parker (TOBEY MAGUIRE) is a high school senior who's been raised by his aunt May (ROSEMARY HARRIS) and uncle Ben (CLIFF ROBERTSON) and is considered something of a geek by other students at his school. That is, except for his friend, Harry Osborn (JAMES FRANCO), and next-door neighbor, Mary Jane Watson (KIRSTEN DUNST), who doesn't realize he's had a crush on her since they were kids.
While on a field trip to a university science department where researchers have created genetically altered spiders, Peter is bit by one, but doesn't think much of it. The next morning, however, he awakens with a new, muscular body and no further need for his glasses. At school, he then discovers that he has incredibly quick reflexes and heightened senses, as well as the ability to shoot webs from his wrists.
At the same time, Harry's wealthy industrialist father, Norman Osborn (WILLEM DAFOE), has decided to test an unproven human performance enhancer on himself so that his company, Oscorp, will win a lucrative contract with the military. Although he's aware of the potential side effects of violence, aggression and insanity, he doesn't realize that it will turn him into a homicidal lunatic. It, of course, does, and gives him a split personality where Norman is unaware of his new and powerful alter ego, the Green Goblin.
Meanwhile, Peter has adapted to his newfound skills - that include swinging through the city from building to building via his webs - and has even adopted the name, "Spider-Man," for a wrestling match to earn some money. Yet, when tragedy strikes his family at the hands of a criminal, Peter decides he must use his powers to fight crime.
From that point on, and after taking a freelance job shooting photographs for Daily Bugle editor J. Jonah Jameson (J.K. SIMMONS) while still trying to get up the nerve to tell Mary Jane how he feels about her, Peter must keep his superhero identity secret all while battling various criminals, including the Green Goblin who sets out to destroy him.