Pelini began his coaching career at the University of Iowa as a graduate assistant for the Iowa Hawkeyes under in 1991. In 1994, Pelini got his first position in the National Football League when he was hired by San Francisco 49ers as a scouting assistant. He was quickly promoted to assistant secondary coach, and by the spring of 1994 he had been promoted again to Defensive Backs Coach. In 1995, in his new position, he coached in his first Super Bowl as the 49ers defeated the San Diego Chargers 49–26 in Super Bowl XXIX.

In 1997, Pelini was hired by New England Patriots, again as defensive backs coach, helping the Patriots reach the playoffs twice during his three years there. In 2000, Pelini became the Linebackers Coach for the Green Bay Packers. Pelini returned to the college ranks in 2003 when he was hired as Defensive Coordinator for the Nebraska Cornhuskers. Pelini was named the interim head coach at the end of the regular season and led the Cornhuskers to a 17-3 win over the Michigan State Spartans in the 2003 Alamo Bowl.

For 2004, Pelini joined the Oklahoma Sooners as co-defensive coordinator. In 2005, Pelini was hired by Louisiana State University Tigers, again as defensive coordinator. His success continued, as LSU was ranked 3rd nationally in overall defense for each of his three years with the Tigers. Pelini was the Head Coach of Nebraska from 2008-2014. In seven seasons at Nebraska, Coach Pelini was 67-27.