Guillen was originally signed as an undrafted amateur free agent with the Pittsburgh Pirates on August 19, 1992. He joined the Pirates' minor league affiliates in 1994 for his first few years of conditioning. He was called up to make his MLB debut with the Pirates on April 1, 1997, joining the Pirates full roster for the 1997 season. He was also seventh in voting for the MLB Rookie of the Year Award. He continued with the Pirates over the next year and a half, playing in almost every regular season game.

On July 23, 1999, Guillen was traded to the Tampa Bay Devil Rays, where he stayed for the next two and a half seasons. Following the 2001 season Guillen signed as a free agent with the Arizona Diamondbacks on December 18, 2001. Just over seven months later the Diamondbacks released him and he signed with the Colorado Rockies on July 29, 2002. Guillen never saw a single game in a Rockies uniform however as he was released just three days later.

Guillen signed as a free agent with the Cincinnati Reds on August 20, 2002, where he played for just short of a year. On July 30, 2003 he was traded to the Oakland Athletics, where he finished the 2003 season. He joined the Athletics for the beginning of the 2004 season, then was suspended for the last two weeks for "innapropriate conduct", stemming from his public comments on his removal from a game late in the season. Following the 2004 season, Guillen was released by the Athletics.

On December 20, 2003, he signed as a free agent with the Anaheim Angels. The following year he was traded to the Washington Nationals on November 19, 2004, where he played through the next two seasons.

Guillen signed as a free agent on December 4, 2006 with the Seattle Mariners. He played 152 games during the 2007 season with the Mariners, finishing with 84 runs, 23 home runs and a 2.90 batting average.

On December 4, 2007, Guillen signed as a free agent with the Kansas City Royals. Just days later he was named in the Mitchell Report, alleging he used steroids. The MLB suspended Guillen for 15 days for violating their drug policy.