The plot of Eyeshield 21 revolves around a physically unassuming and non-assertive boy named Sena Kobayakawa who enters the high school of his choice, Deimon Private Senior High School, where his childhood friend Mamori Anezaki, one year older than Sena, already attends. His only remarkable physical abilities are his running speed and dodging techniques, the result of a school life spent complying with the demands of bullies, quickly catch the eye of the high school's American football team captain Yoichi Hiruma who then forces Sena to join the team (Deimon Devil Bats) as the running back.
To protect his identity (on the premise that other school sports teams will try to recruit Sena upon discovering his athletic skill) Sena is forced to publicly assume the role of team secretary, and enter the field wearing a helmet equipped with a green-tinted eyeshield in order to hide his features under the pseudonym of "Eyeshield 21." The makeshift team initially takes part in the Spring football tournament (at the beginning of the Japanese school year) hoping to win with through the strength of their new "secret weapon." But the extremely weak team is eliminated early on by the Ojo White Knights, a powerhouse football team. Others see the skill of the current White Knights as having declined from the previous "Golden Generation" but the team members are trying to prove that they have the ability to surpass their predecessors. It is in this game that Sena meets the man he will acknowledge as his ultimate rival, Seijuro Shin, a talented linebacker.
After Deimon's defeat, the Spring tournament is revealed as a secondary in importance to the Fall tournament, where the teams compete for the chance to play in the "Christmas Bowl," the high school football league championship. Hiruma, Kurita and Sena regroup and slowly build a real team from other misfits and students looking to define themselves, with the goal of reaching the championship. The series follows the building and growth of the Deimon Devil Bats and its members, as well as various rival teams encountered through their games and tournaments, as they all strive to achieve their goal of playing in the Christmas Bowl.