Gardner attended walk-on tryouts for the baseball team at the College of Charleston in 2001. Gardner became a three-year starter for the College of Charleston Cougars. Gardner had a .397 batting average in 2004, his junior year, but he was not selected in the 2004 MLB draft. In 2005, his senior year, he batted .447, tied for the most hits in college baseball with 122, established a Cougars record with 85 runs scored, and led the Southern Conference with 38 stolen bases.

Gardner finished the 2005 season in the New York–Penn League season ranking 5th in at bats (with 282), 2nd in runs (62), and 5th in stolen bases (19). He was a Florida State League All Star in 2006, batting .323 in 63 games with 22 RBIs with the Tampa Yankees. He was third in the Florida State League in batting average and led the league in stolen bases with 30. Gardner also was second in the league in walks with 47.

In 2007, he played 54 games for the Double-A Trenton Thunder, though he missed time with a broken bone in his hand. In 203 at bats, he stole 18 bases (tied for 5th in the league; while being caught 4 times), hit five triples, and batted .300 with a .392 OBP, before being promoted to the Triple-A Scranton/Wilkes-Barre Yankees. There, in 45 games, he batted .260 with a .343 OBP, and stole 21 bases while being caught only three times.

In fall 2007, he played in 26 games in the Arizona Fall League, leading it in runs (27) and stolen bases (16), while being caught stealing only once. He batted .343 (5th in the league) with a .433 OBP (3rd) and was third in the league in walks with 17. In 2007, he was the 12th-best prospect in the Yankees minor league system according to Baseball America.

Playing for Scranton/Wilkes-Barre in 2008, in 94 games Gardner was second in the International League with a .414 on-base percentage, 70 walks, 11 triples, and was 6th in the IL with 37 stolen bases, while being caught only nine times.

Through 2008 in the minor leagues, he had a .291 batting average and a .389 OBP. He had stolen 153 bases, being caught only 31 times (an 83% success rate).

On June 30, 2008, Gardner was called up and made his major league debut, batting leadoff and going 0-for-3 with a stolen base. On July 2, he earned both his first hit and first RBI, off fellow rookie Texas Rangers relief pitcher Warner Madrigal in the seventh inning. Gardner went on to steal second and eventually score in that inning. On July 26, 2008, Gardner was optioned back to AAA after the acquisition of Xavier Nady in order to continue to receive playing time. The Yankees again recalled Gardner on August 15.

On September 21, 2008, Gardner scored the final run of Major League Baseball in Yankee Stadium history as a pinch runner for Jason Giambi, scoring on a sacrifice fly by Robinson Canó in the seventh inning of an eventual 7–3 win for the Yankees over the Baltimore Orioles. Gardner finished his rookie season playing 42 games, batting .228 with 16 RBI and 13 stolen bases.

On December 7, 2010, Gardner underwent surgery to get rid of inflamed tissue in his wrist, with Yankees general manager Brian Cashman stating that he would be back in time for spring training.

Gardner is considered one of the fastest players in Major League Baseball. He is best known for stealing bases and being very disciplined at the plate. He makes contact with 93% of his swings, third best in the American League. In 2010 and 2014, Gardner saw more pitches per at-bat than any other player in the American League.

Gardner and the Yankees agreed on a $2.8 million contract for the 2012 season, avoiding arbitration. Gardner experienced an elbow injury in 2012 and was expected to return in August. On July 16, Gardner suffered a setback and his chances of missing the entire season increased. On July 24, Gardner underwent right elbow surgery, performed by Christopher S. Ahmad, to remove a bone spur and inflamed tissue. He was reactivated on September 25 after Steve Pearce and Justin Thomas were designated for assignment. Gardner played in 16 games during 2012, batting .323 with two stolen bases and three RBI.

On August 11, 2013, Gardner hit his first career walk-off home run with two outs in the bottom of the 9th inning off of Detroit Tigers reliever José Veras. The home run sealed a 5–4 victory for the Yankees, and helped give support to a struggling Mariano Rivera, who blew three consecutive save opportunities for the only time in his career. In the same series against Detroit two days earlier, Gardner made his first bailout of Rivera’s pitching mishap by hitting a walk-off single past a diving Miguel Cabrera to give the Yankees a 4–3 win. According to the Elias Sports Bureau, Gardner became the first Yankee since Claudell Washington in 1988 to have two walk-off hits in a span of three or fewer games. In 2013, Gardner played 145 games batting .273 with 8 home runs, 33 doubles, an American League leading 10 triples, 52 RBI, and 24 stolen bases.

On February 23, 2014, the Yankees and Gardner agreed to a four-year, $52 million extension to begin in 2015. On April 23, 2014, Gardner made his first career start at right field. On July 28, 2014, Gardner recorded his first career multi-homer game where he hit 2 home runs off of Texas Rangers pitcher Yu Darvish. He was named AL Player of the Week on August 4. On September 21, Gardner recorded the 15,000th home run of the Yankees franchise off the Blue Jays’ Drew Hutchison. Gardner struggled in September due to an injury, finishing 12-for-72 (.167), dampening his strong 2014 offensive season. Gardner changed his approach to hitting, relying less on his declining speed and altering his swing to develop more power. For the 2014 season, Gardner played in 148 games, batting .256 with a career-high 17 home runs, 58 RBI, and 21 stolen bases, and had the highest number of pitches per plate appearance in the major leagues (4.44).

Gardner was named AL Player of the Week for the week ending June 28, 2015. He was one of the five candidates chosen for the All-Star Final Vote for the 2015 MLB All-Star Game, but was later removed from the ballot after being chosen to replace the injured Alex Gordon on the All-Star team. Gardner’s hot first half would not last the whole season; after batting .302 up to the All-Star break, he struggled for the rest of the year, hitting .206 in the second half of the season. After going 0–4 with 3 strikeouts in the Yankees’ 3–0 loss to the Houston Astros in the 2015 AL Wild Card Game, Gardner received a loud roar of boos from fans at Yankee Stadium.

In 2015, Gardner continued his hitting approach from the prior season, batting .259 with 16 home runs, 66 RBI, and 20 stolen bases.

Gardner was moved into the Yankees’ leadoff spot midseason after traditionally hitting behind Jacoby Ellsbury during Ellsbury’s tenure as a Yankee. On April 23, Gardner hit a walk-off home run against the Tampa Bay Rays. Gardner’s on-base percentage in 2016 was .351, his highest in a full season since 2010, and his 70 walks were his most since that same season. However, his .713 OPS was tied with 2011 for his lowest career mark in a full season. For the 2016 season, Gardner hit .261 with 7 home runs, 41 RBI, and 16 stolen bases. His home run and RBI count were the lowest in a full season since 2011, while he recorded the lowest stolen base count of his career in a full season. He won the Gold Glove Award for American League left fielders.

On April 12, 2017, Gardner collided with Rickie Weeks at first base, and both players left the game. Gardner did not sustain a serious injury and came away only with a bruised jaw and strained neck. The Yankees listed him as day-to-day. On April 29, Gardner hit 2 home runs in a 12–4 win over the Baltimore Orioles. On May 2, Gardner had another two home run game in an 11–5 win over the Toronto Blue Jays. On May 5, with the Yankees down to their final strike, Gardner hit a go-ahead three-run home run off of Hector Rondon, which proved to be the game winner as the Yankees won 3-2 over the Chicago Cubs. On June 1, Gardner collected the 1,000th hit of his career off Marco Estrada of the Toronto Blue Jays. On June 30, Gardner went 3-for-5 with a grand slam and 6 RBIs, tying his career high, while falling a triple short of the cycle. On July 27, Gardner hit his 18th home run of the season, a walk-off against the Tampa Bay Rays, setting a new career high for single-season home runs. The next day, Gardner started the game with a leadoff home run. Gardner hit a walk-off single on July 29 with the bases loaded in the bottom of the ninth. Gardner hit a career-high 21 home runs, recording 23 steals and a .264 average as the Yankees made the playoffs and finished one game shy of the World Series. Gardner had a productive postseason, batting .286 with 2 RBI in the American League Division Series versus the Cleveland Indians. He was listed as a Gold Glove finalist, but ultimately lost to Alex Gordon of the Kansas City Royals.

Gardner entered the 2018 season as a member of a crowded outfield. However, due to injuries to Jacoby Ellsbury, (who missed the entire 2018 season with various ailments), Clint Frazier, who suffered a concussion in spring training that sidelined him for most of the season, and Aaron Judge (who missed two months with a wrist injury), Gardner saw more playing time than originally expected, appearing in 140 games. He finished the season batting .236 (his worst average since 2008), with 12 home runs and 45 RBIs. On October 31, 2018, the Yankees announced they had declined his $12.5 million option for 2019, and instead re-signed him to a one-year, $7.5 million contract for the 2019 season.

On April 17, 2019, Gardner hit his 100th career home run—a grand slam off Ryan Brasier—to give the Yankees a go-ahead 5–3 lead over the Boston Red Sox in what would be the game-deciding hit. He became one of two players in Yankees history (alongside Derek Jeter) to hit at least 100 home runs and steal at least 250 bases. On July 26, the Yankees placed Gardner on the 10-day injured list (retroactive to July 22nd) for left knee inflammation stemming from an injury he had suffered the previous week, putting him on the injured list for only the third time in his major league career. He was recalled from the injured list on August 2.

Gardner hit .251 with a career-high 28 home runs and 74 RBIs in 2019. He finished third in Gold Glove voting.

After the 2019 season, Gardner became a free agent. The Yankees re-signed Gardner to a one-year contract with an option for the 2021 season.

In the shortened 2020 season, Gardner, again in a fourth-outfielder role, played in 49 games, hitting .223 with five home runs and 15 RBIs. On October 29, 2020, the Yankees declined their 2021 $10 million option on Gardner, making him a free agent.