Peter is bitten by a radioactive spider (erroneously classified as an insect in the panel) at a science exhibit and "acquires the agility and proportionate strength of an arachnid." Along with super strength, he gains the ability to adhere to walls and ceilings. Through his native knack for science, he develops a gadget that lets him fire adhesive webbing of his own design through small, wrist-mounted barrels. Initially seeking to capitalize on his new abilities, he dons a costume and, as "Spider-Man", becomes a novelty television star. However, "He blithely ignores the chance to stop a fleeing thief, his indifference ironically catches up with him when the same criminal later robs and kills his Uncle Ben." Spider-Man tracks and subdues the killer and learns, in the story's next-to-last caption, "With great power there must also come—great responsibility!"

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Despite his superpowers, Parker struggles to help his widowed aunt pay rent, is taunted by his peers—particularly football star Flash Thompson—and, as Spider-Man, engenders the editorial wrath of newspaper publisher J. Jonah Jameson. As he battles his enemies for the first time, Parker finds juggling his personal life and costumed adventures difficult. In time, Peter graduates from high school, and enrolls at Empire State University, where he meets roommate and best friend Harry Osborn, and girlfriend Gwen Stacy, and Aunt May introduces him to Mary Jane Watson. As Peter deals with Harry's drug problems, and Harry's father is revealed to be Spider-Man's nemesis the Green Goblin, Peter even attempts to give up his costumed identity for a while. Gwen Stacy's father, New York City Police detective captain George Stacy is accidentally killed during a battle between Spider-Man and Doctor Octopus. In the course of his adventures Spider-Man has made a wide variety of friends and contacts within the superhero community, who often come to his aid when he faces problems that he cannot solve on his own.

The Green Goblin throws Gwen Stacy from a tower of either the Brooklyn Bridge (as depicted in the art) or the George Washington Bridge (as given in the text). She dies during Spider-Man's rescue attempt; a note on the letters states: "It saddens us to say that the whiplash effect she underwent when Spidey's webbing stopped her so suddenly was, in fact, what killed her." The following issue, the Goblin appears to kill himself accidentally in the ensuing battle with Spider-Man.

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Working through his grief, Parker eventually develops tentative feelings toward Watson, and the two "become confidants rather than lovers".A romantic relationship eventually develops, with Parker proposing to her, and being turned down later. Parker went on to graduate from college,and becomes involved with the shy Debra Whitman and the extroverted, flirtatious costumed thief Felicia Hardy, the Black Cat. From 1984 to 1988, Spider-Man wore a black costume with a white spider design on his chest. The new costume originated in the Secret Wars limited series, on an alien planet where Spider-Man participates in a battle between Earth's major superheroes and villains. He continues wearing the costume when he returns from the Secret Wars, starting in The Amazing Spider-Man. Not unexpectedly, the change to a longstanding character's iconic design met with controversy, "with many hardcore comics fans decrying it as tantamount to sacrilege. Spider-Man's traditional red and blue costume was iconic, they argued, on par with those of his D.C. rivals Superman and Batman." The creators then revealed the costume was an alien symbiote which Spider-Man is able to reject after a difficult struggle, though the symbiote returns several times as Venom for revenge.

Parker proposes to Watson a second time in The Amazing Spider-Man, and she accepts, with the wedding taking place in The Amazing Spider-Man Annual.

Peter becomes convinced that Ben Reilly, the Scarlet Spider (a clone of Peter created by his college professor Miles Warren) is the real Peter Parker, and that he, Peter, is the clone. Peter gives up the Spider-Man identity to Reilly for a time, until Reilly is killed by the returning Green Goblin and revealed to be the clone after all.He develops additional spider-like abilities including biological web-shooters, toxic stingers that extend from his forearms, the ability to stick individuals to his back, enhanced Spider-sense and night vision, and increased strength and speed. Peter later becomes a member of the New Avengers, and reveals his civilian identity to the world, furthering his already numerous problems. His marriage to Mary Jane and public unmasking are later erased in another controversial storyline "One More Day", in a Faustian bargain with the demon Mephisto, resulting in several adjustments to the timeline, such as the resurrection of Harry Osborn, the erasure of Parker's marriage, and the return of his traditional tools and powers.