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 who attended the year before. His only remarkable physical abilities are his running speed, and intense agility; The result of a school life spent complying with the demands of bullies, it was revealed later a friend taught him how to run quickly as a base. His abilities quickly catches the eye of the high school's American football team captain Yoichi Hiruma who forces Sena to join the Deimon Devil Bats as its running back.
To protect his identity to prevent other teams in the school to recruit him for his speed, 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 hoping to win with through the strength of their new "secret weapon." However, the extremely weak team is eliminated early on by the Ojo White Knights, a powerhouse football team that focuses on defense.
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, such as TarÅ Raimon a baseball player who can only catch, and the the three Ha-Ha Brothers. Other characters slowly join the team as the series follows the building and growth of the Deimon Devil Bats and its members as well as various rival teams as they all strive to achieve their goal of playing in the Christmas Bowl.
Following their victory at the Christmas Bowl, Japan begins to gather the best football players to form a team that will represent Japan at the American Football Youth World Championship where an MVP will be rewarded a NFL contract and $3 Million. Team Japan manages to make it to the final against Team America. The result is that a final touchdown by Sena at the buzzer ties the score and as the people putting on the Tournament hadn't planned that anyone would put up a fight against the US, both teams were declared the winner; however Panther of Team America is chosen to be the MVP.
The series concludes with Sena becoming the captain of the Devil Bats when Hiruma and Kurita go off to college. In his final year of high school, Sena is invited to Notre Dame High School by Clifford, therefore bringing Hiruma's proclamation at the beginning of the series true: the famous Eyeshield 21 of Notre Dame High has been born. All of the main characters are shown in the final chapter to be in college or playing amateur-league football while maintaining a job.