Pelini was raised in Youngstown, Ohio, a former center of steel production with a strong athletic tradition. He was nicknamed "Bo" after former Cleveland Browns running back Bo Scott. After graduating from Youngstown Cardinal Mooney High School (the same high school as Oklahoma Sooners Head Coach Bob Stoops), he went on to play free safety for Ohio State University Buckeyes under Earle Bruce and later John Cooper from 1987 to 1990, as a starter in his last two years. Pelini served as a team co-captain in his senior year, along with Vinnie Clark, Jeff Graham and Greg Frey. He earned his bachelor’s degree in business marketing from Ohio State University in 1990 while playing for the Buckeyes.