Peter Parker is a shy, nerdy senior in high school. He is a brilliant scientist for his age, and lives with his elderly Uncle Ben and Aunt May, who have raised him, presumably since his parents were killed. In the first scene, Peter sees and narrates his love for Mary Jane Watson, the literal ‘girl next door’ to Peter. She is pretty and popular, the kind of girl Peter could never dream of having. Peter’s best friend is Harry Osborn, and along with being friends, the two help each other with school (usually Peter assisting Harry with math and science). Harry’s father is the workaholic Norman Osborn, president of the Oscorp Manufacturing Corporation, a primary supplier of advanced weapons to the United States Army.
The first few scenes introduce us to the protagonist, Peter Parker, as well as his best friend Harry Osborn and crush Mary Jane Watson. Peter is going on a field trip with his class to a highly respected genetics lab. During the tour, someone notes a spider is missing, but the tour guide dismisses the observation, saying it’s probably in testing. Peter is then bitten by the spider (although he does not know it is the test spider) and crushes it.
At the lab of his company, Norman Osborn shows off his latest invention to an Army general: an advanced flying glider with various weapons that a man can ride. He then turns to what the general seems most interested in: a chemical that enhances physical human attributes to super-human levels. He tells the general the formula is ready, but his bumbling partner Dr. Stromm pipes in that it is not, as it has shown an increase in aggression and irrational violence in all test subjects. Osborn is angered by the undermining, and the general dismisses both men as well as the formula, informing Osborn of the Army’s intent to pull all funding and transfer all contracts to Oscorp’s biggest competitor.
Back home, Ben and May are discussing money issues when Peter arrives and goes upstairs, claiming he feels ill. He passes out on the floor and has confusing dreams. We zoom in on his DNA, seeing it transform to that of a spider.
Meanwhile, back at Oscorp, Osborn is proceeding to human testing, but without approval from anyone he has only one test subject: himself. He proceeds to ignore Stromm’s protests. When the test goes awry, he loses his mind and kills Stromm in a fit of rage.
The next day, Parker rushes out of to the bus and finds that, as he bangs on the side, the banner sticks to his unclenched hand. Later, during lunch of the same day, he sits down at a table in the cafeteria. Watson passes him, and in Parker's mind he sees her slipping on a puddle, falling back and throwing her lunch into the air. Parker reacts almost before it happens and catches her, then her tray, then all of her lunch items on the tray. They have a short conversation, and then Mary Jane goes back to her popular friends. Parker tries to put down a fork, but finds that it too is stuck to his hand. He pulls it off with his other hand and finds a strand of webbing shoots out of his wrist. It snags a tray of food, and Parker jerks it, trying to detach the webbing. It instead pulls the whole tray, which lands on Flash and covers him in food. Parker quickly gets up and exits, only to drag the tray behind him by his webbing. Flash looks around for whoever hit him with a lunch and sees Parker exit.
The two fight outside and Parker easily bests him, watching the punches fly by his head. Parker flees his school and finds that he can crawl up walls and has incredible agility, as well as an organic webbing he can use to swing from building to building. Later, Parker takes out the trash and has a conversation with MJ; it is cut short, however, by her boyfriend driving up in his fancy new sports car. The next few scenes show Parker practicing his new abilities and designing a basic costume. He also looks in the paper and sees several car ads. They are all very expensive. He then sees an ad for a fight club, offering $3,000 for anyone who can last three minutes with the champion.
Parker has a heated argument with Uncle Ben on his way to the fight club. After he gets there, he easily bests the champion in less than two minutes. When he goes to collect his winnings, the promoter refuses to pay him. As Parker turns to leave, the promoter is robbed, and Parker lets the thief walk right past him in revenge. Later, Parker sees Uncle Ben in the middle of a crowd. Ben has been shot. Parker holds Ben until he dies, then overhears two cops saying they’ve found the killer and are chasing him. Parker quickly joins the chase and catches the guy, recognizing him as the thief from the fight club. Parker realizes if he’d done the right thing Ben would still be alive. During their fight, the thief trips and falls out of a window, to his death.
Later in the night, the general that fired Osborn and Oscorp is at a demonstration at the competitor’s facility. A masked and cackling figure comes out of the sky and kills them all.
Soon after graduation, Parker moves into an apartment with Harry and designs a new Spider-Man outfit. He assumes his identity as the protector of New York. He begins taking pictures of himself as Spidey for the Daily Bugle, only to find their unscrupulous editor J. Jonah Jameson is using the pictures and headlines to paint Spider-Man as a menace to the city. Also, Parker discovers that Harry and MJ are dating, but does not tell Harry he knows.
Osborn’s company decides to sell out to their competitor, but also fires him. At a large parade a day or so after, all the members of the Oscorp board, as well as Parker, MJ and Harry, are attending. During the parade, the masked figure kills all the board members and attacks Mary Jane, but is defeated by Spider-Man. The villain slinks off to lick his wounds.
The next day, Parker goes to deliver his pictures of the festival and of the fight to Jameson, who has just named the villain the Green Goblin. Just as Parker leaves, the Goblin attacks, trying to find a link to Spider-Man. Spidey shows up to save Jameson, but is captured by the Goblin, who tries to talk him into becoming his partner. Spider-Man refuses and the Goblin lets him go, telling him the city will turn against him sooner or later.
Soon after, MJ is attacked and saved by Spider-Man, and the two share a passionate kiss. Spider-Man then goes to save someone from a burning building, but finds it is the Goblin, who attacks and injures him. Both escape and switch into their secret identities. They then both go to a dinner with the Parkers, Osborns and Watson all in attendance. During the meal, Osborn sees Parker's scratch and realizes he is Spider-Man, but says nothing. Later that night, he and the Goblin argue with each other about how to deal with Spider-Man. In the end, they decide to kill those close to him and then Spidey himself. Goblin attacks May but does no permanent damage, and then kidnaps MJ. Spider-Man finds him and barely saves MJ and a few civilians the Goblin also endangered. The Goblin then drags Parker off to a new location where the two fight; Parker emerges victorious. Osborn reveals his true identity and presses a button on his suit, which positions his glider silently behind Spider-Man. He then hits another button and it flies forward, but Parker dodges and it kills the Goblin instead. His last words are a request that Parker not tell Harry about his alter ego.
Parker delivers Osborn’s body to the Osborn residence, but Harry catches him. However, he only sees the Spider-Man outfit and vows to make Spider-Man pay, thinking he killed his father. The last scene of the movie is the funeral, at which MJ tells Parker she loves him. However, he has realized that if any of his enemies find out his identity, being close is only endagering her. Parker tells MJ he does not love her, so he can continue being Spider-Man without endangering her.