At the age of 17, Tatís was signed as an amateur free agent by Omar Minaya and the Texas Rangers on August 25, 1992. He played his first game in Major League Baseball with the Rangers, at third base, almost five years later on July 26, 1997, and went on to play 60 games with the Rangers in his rookie season. At the trade deadline on July 31, 1998, the Rangers traded Tatís along with Darren Oliver and Mark Little to the St. Louis Cardinals for Royce Clayton and Todd Stottlemyre.

Tatís had the best season of his career in 1999 with the St. Louis Cardinals. He hit 34 home runs with 107 RBIs and 21 stolen bases, with a .298 batting average. On April 23, 1999, Tatís made baseball history when he hit two grand slams in one inning. He is the only batter in MLB history to accomplish this feat. Tatís hit both of his grand slams against starting pitcher Chan Ho Park of the Los Angeles Dodgers. With these two grand slams Tatís also set a Major League record with eight runs batted in during a single inning.

After playing in only 96 games for the Cardinals in 2000 because of an injury, Tatís was traded to the Montreal Expos along with Britt Reames for Dustin Hermanson and Steve Kline. Tatís played in just 208 games out of a possible 486 over three seasons with the Expos because of an assortment of physical injuries.

The Tampa Bay Devil Rays invited Tatís to spring training in 2004, but he did not make the team and was released. Tatís next did not play professional baseball for two seasons and resided in the Dominican Republic.

The Baltimore Orioles signed Tatís to a minor-league contract on November 25, 2005. Tatís returned to baseball in order to raise money to build a church. He played most of the season for the AAA baseball, the Ottawa Lynx, eventually playing in 28 games for the Orioles after being called up on July 21, 2006.

In 2007, Tatís was invited to spring training with the Los Angeles Dodgers. After being assigned to minor-league baseball camp, Tatís was granted his request to be released from his contract on March 14. Just nine days later, he signed a minor-league contract with the New York Mets, and spent the 2007 season with its AAA affiliate, the New Orleans Zephyrs.

On May 11, 2008, Tatís was called up from the Zephyrs to replace Ángel Pagán. Tatís had started to play the outfield in the minor leagues in order to become a more diversified player.

On September 16, 2008, Tatís separated his shoulder after diving for a fly ball in a game against the Washington Nationals. The Mets team physician diagnosed the injury as being a Grade III separation, but Tatís missed the rest of the 2008 regular season. Despite missing the end of the season, on October 23, 2008, Tatís received the Sporting News Comeback Player of the Year Award for the National League.

Tatís played intermittently for the Mets in 2009 and 2010. His last Major League game was on July 4, 2010. He was also the latest New York Met to wear uniform number 17, which was taken out of circulation as a mark of respect for Keith Hernandez, who had worn it as a member of the Mets from 1983 to 1989.

On October 5, 2014, Tatís announced his retirement as a player. For his career, he batted .265 with 113 home runs and 448 RBIs.

In January 2018, the Boston Red Sox announced that Tatís had joined their minor league organization as manager of one of their two rookie-level Dominican Summer League Red Sox teams.

Tatís’s son, Fernando Tatís Jr., is a shortstop for the San Diego Padres. His younger son Elijah is also a baseball player and was signed by the Chicago White Sox in 2019.