DeJesus was raised in Manalapan Township, New Jersey, and played high school baseball at Manalapan High School. He was drafted out of high school by the New York Mets in the 43rd round of the 1997 Major League Baseball Draft. DeJesus did not sign with the Mets, opting instead to attend Rutgers University. Three years later, he was drafted in the fourth round of the 2000 Major League Baseball Draft by the Kansas City Royals.
DeJesus made his Major League debut on September 2, 2003. After Carlos Beltrán was traded to the Houston Astros in 2004, DeJesus became the starting center fielder for the Royals. He finished sixth in the 2004 Rookie of the Year voting. On March 9, 2006, DeJesus signed a five-year, $13.8 million contract extension with the Royals through 2010, with a club option for the 2011 season. On June 15, 2008, De Jesus hit the first grand slam of his career. This prompted the game caller to remark, "He smashed the living DeJesus out of that ball!"
In 2010, DeJesus was in threat of being traded along with his fellow starting outfielders, Scott Podsednik and José Guillén, who were both traded. DeJesus, on the other hand, tore a tendon on his right thumb crashing into a fence in Yankee Stadium in July and had season ending surgery. DeJesus was hoping to return to the team in September, but instead was moved to the 60-Day DL. He finished the 2010 season batting .318 with 5 home runs and 37 RBIs in 352 at-bats.
On October 2, 2010, the Royals picked up his $6 million club option for 2011.
On November 11, 2010, DeJesus was traded to the Oakland Athletics for pitcher Vin Mazzaro and minor league pitcher Justin Marks.
On November 30, 2011, DeJesus signed a two-year deal worth $10 million, to be the primary right fielder for the Chicago Cubs.
In 2013, due to the signing of Nate Schierholtz, DeJesus moved to center field for the 2013 season. While in Chicago, David quickly became a fan favorite for the Cubs, due to his outstanding work ethic and positive attitude.
On August 19, 2013, DeJesus was traded to the Washington Nationals for a player to be named later. He appeared in three games for the Nationals, going 0-for-3.
On August 23, the Nationals traded DeJesus to the Tampa Bay Rays for future considerations. DeJesus got off to a hot start with the Rays culminating in a walk-off single in the 18th inning against the Baltimore Orioles scoring Desmond Jennings.
On November 6, 2013, DeJesus re-signed with the Rays, inking a two-year, $10.5 million deal. The Rays, without a true designated hitter, put DeJesus in that role in 2014, where he appeared the most. On June 19, DeJesus was placed on the disabled list with a left hand fracture on a check swing the previous day. He was hitting .269 with 5 HR and 17 RBI in 62 games before the injury. He was leading the team with a .367 OBP and a .440 SLG.
On July 28, 2015, DeJesus was traded to the Los Angeles Angels of Anaheim for minor league pitcher Eduar Lopez.
DeJesus is married to former model and Amazing Race 23 contestant Kim DeJesus.