National College Players Association President Ramogi Huma filed the petition with the National Labor Relations Board, along with cards signed by an undisclosed number of players indicating they wish to be represented by the union, ESPN reported.
Human -- who played football at UCLA and helped form the NCPA in 2001 -- said the unionization movement has the support of the United Steelworkers union.
"This is about finally giving college athletes a seat at the table," he said. "Athletes deserve an equal voice when it comes to their physical, academic and financial protections."
NCAA Chief Legal Officer Donald Remy issued a statement saying "student-athletes are not employees within any definition of the National Labor Relations Act" so there is no employment relationship between the "NCAA, its affiliated institutions or student-athletes," ESPN said.
"This union-backed attempt to turn student-athletes into employees undermines the purpose of college: an education," Remy said.
The statement said student-athletes' participation in college sports is "voluntary."
Northwestern quarterback Kain Colter -- who has become a high-profile advocate for unionization -- said filing petitions for representation is not an indication the school mistreats players.
"We love Northwestern," he said. "The school is just playing by the rules of their governing body, the NCAA. We're interested in trying to help all players -- at USC, Stanford, Oklahoma State, everywhere. It's about protecting them and future generations to come.
"Right now the NCAA is like a dictatorship. No one represents us in negotiations. The only way things are going to change is if players have a union."