Peter Parker's story begins with him chasing down his school bus, which is clear to be a regular occurrence and just one of the many forms of bullying Peter is subject to in his high school career. His long-time crush and neighbor since age six, Mary Jane, intervenes and has the driver stop the bus to let him on, and it's made apparent that Peter is also rather socially isolated. The bus arrives at it's destination- a field trip at The Columbia University science department.

The camera cuts to Harry Osborn, who is arguing with his father, Norman Osborn. Here we learn two key things about Harry's character: one, that Harry is insecure about his wealth and the way it causes him to struggle with connecting to his peers, and two, that Harry has flunked out of every private school in New York that Norman has tried to send him to. As Harry gets out of the car, he immediately goes to find Peter, but is followed by his father. As a result, Harry ends up introducing Peter and Norman to each other.

As the field trip commences, Peter drops some fun facts about the lab they're in to Harry, who in turn, insists that Peter go talk to Mary Jane. Peter declines, telling Harry to go talk to her instead. It's at this point that the group starts to learn about Oscorp's experiments with genetic mutation, and how they've created fifteen brand-new species of "super-spiders" by cross-breeding different spider subspecies. One of Peter's classmates points out that one of the spiders is missing, but the person touring the group brushes it off as the researchers actively working on it. As the group moves on to the next exhibit, their teacher chides Harry for talking as Peter gets Mary Jane to pose for school newspaper photos. After Peter snaps his pictures, Mary Jane walks off to join the group and in the few moments that Peter is alone and unwatched, the missing fifteenth spider drops from the ceiling and bites his hand. The pan between the spider crawling away and Peter's face tells us that he knows exactly what bit him, and it's fair to assume that he thinks he's going to die.

Meanwhile, at Oscorp Industries, Norman Osborn recieves an unexpected visit from his board of directors, as well as General Slocum of the United States Military. They are there to check in on the progress of Norman's eugenics experiment, the human enhancement serum. They inform him that the stakes are high- if they can't have the formula ready for human trials by the following week or they will cut their funding to and contract with Oscorp. Norman claims that it's ready for human testing and that everyone on his lab team agrees except for Dr. Stromm, but when prompted, Dr. Stromm hesitantly says it needs to be taken back to formula.

At the end of Peter's school day, we get introduced to Aunt May and Uncle Ben with an exposee of the Parker's financial situation. Uncle Ben has been put out of a job after thirty five years of work as a senior electrician at the city plant, and all the job openings available in the newspaper are for computer jobs that he lacks the skillset for. He considers himself outdated for the current job market. As Aunt May reassures him, Peter comes home and rushes past them up to his room, already feeling the effects of the spider bite. The early symptoms appear to be dizziness, blurry vision, chills, cold sweats, confusion, heightened sensitivity to light and sound, headaches, and sleepiness. Upon making it to his bedroom, Peter locks the door, rolls over his bed onto the floor, pulls his blanket down, and falls asleep.

As Peter sleeps, his body undergoes an overnight transformation. His visual impairment is cured, his muscles develop as though he's been working out for months, and he develops a white scar in the shape of a star on his right wrist below his thumb. At the same time as his transformation, Norman is with Dr. Stromm in the Oscorp lab to volunteer himself as the first human test subject for the human enhancement serum. As the serum is administered, Norman flatlines briefly, rescusitated by the serum effectively transforming his body into something superhuman. He turns on Dr. Stromm after screaming at him about his disapproval of the serum, murders him and flies off into the night with the glider that Oscorp has developed.

The next morning when Peter wakes up, he notices the changes to his body immediately- and is not upset in the least about them. As he eats breakfast with Aunt May and Uncle Ben, Uncle Ben reminds him that they'll be painting the kitchen that night, so he'll have to come directly home from school. Over at the Osborn's penthouse, Harry finds his father passed out on the floor of his study. Moments later, Norman's secretary rushes in to inform him that the glider and Green Goblin armor have been stolen and that Dr. Stromm has been murdered. Norman has no recollection of his actions from the night before.

On Peter's way to school, he begins to notice that something more than just his sight and appearance may have changed. As his usual chase down of the bus begins, the poster on the side of the bus gets stuck to his hand and rips off with no feasible explanation. During his lunch period, he intuitively realizes that Mary Jane is going to fall and drop her lunch tray just moments before it happens and is able to catch her. After sitting down at his table, his lunch tray gets stuck to his hand and by way of pure accident, he flings food onto his long time bully and Mary Jane's boyfriend, Flash Thompson. On his way to leave the cafeteria, he accidentally shoots a web from his wrist, dragging his lunch tray behind him.

Flash follows and starts attempting to intimidate Peter. As a crowd forms around them, Flash throws a punch and time seems to slow for Peter, who is able to dodge it with ease. With both of them taken aback and their classmates stunned, the dodge leaves Flash frustrated and embarrased, and he tries another- only for Peter to dodge again. After a few more times dodging Flash's blows, Peter delivers one of his own with a strength that leaves him shocked. As the crowd clears, Harry expresses his amazement and admiration for Peter.

After school, Peter avoids going home in favor of investigating and testing out his newfound powers. He realizes that he's able to stick to and climb up walls and jump a great distance, easily hopping over gaps between building rooftops. He also realizes that he's able to shoot webs from his wrists with the correct hand motion. These webs easily support his weight and more, and allow him to swing from building to building and navigate the city from up high. By the time he's done experimenting, it's dark, and when he returns home, there's a note waiting for him directing him to dinner in the fridge.

After eating his dinner, Peter takes out the trash. While outside, he overhears a fight between Mary Jane and her father, and shortly thereafter, Mary Jane steps outside. While she's initially on the offensive, demandingly asking Peter if he heard what happened inside, but after a reassuring response from Peter her demeanor softens. They make short conversation about post graduation plans.