Inspired by the popular Marvel comic book series, Peter Parker, his best friend Harry Osborn, and Peter's secret crush Mary Jane Watson visit a genetics laboratory at Columbia University with their high school class. While taking photos in the laboratory, Peter is bitten on the hand by a genetically engineered "super spider". Feeling unwell, he passes out shortly after arriving home.

Meanwhile, scientist and owner of Oscorp Norman Osborn, Harry's father, is attempting to preserve his company's military contract, knowing that its loss will mean the end of his business. He experiments on himself with his company's new, but unstable, performance-enhancing chemical vapor which increases his speed, strength, and stamina. However, it also causes him to become insane and he kills his assistant, Mendel Stromm.

The next morning, Peter wakes to find that his previously impaired vision has improved and that his body has metamorphosized into a more muscular physique. At school, he finds himself producing webbing and having the quick reflexes to avoid being injured in a fight with bully Flash Thompson. Peter escapes from the school and realizes that he has acquired spider-like abilities from the spider bite. He quickly learns to scale walls, long jump across building rooftops, and swing via webs from his wrists.

He decides to test out his powers by winning a wrestling match against Bonesaw McGraw to earn $3,000 for a car to impress Mary Jane by donning a costume he made for himself. After the match ended, the manage didn't pay him enough because he pinned 3 minutes in the ring in 2. A robbery occurred in the arena and Peter didn't get him because he cares about being number one until later that night outside Manhattan his Uncle Ben was shot and murdered by a carjacker and Peter sought revenge on the one who did it; it was the same robber who ran passed him from the arena. He then realized Uncle Ben's wise words that with great power comes great responsibility.