The Arizona Diamondbacks selected Upton with the first overall selection in the 2005 MLB draft.

On August 2, 2007, Upton was called up to the big league team at the age of only 19, following an injury to everyday right fielder Carlos Quentin. When he was called up, the Diamondbacks had star rookie Chris Young at center field, so Upton shifted to right.

Upton recorded his first career major league hit and run scored on August 4, 2007, in his third major league game. His first career RBI came the next day, and his first career home run two days after that. He almost became the youngest player ever to hit for the cycle on that day, falling just a single shy of the milestone.

He finished his rookie campaign with 2 home runs, 11 RBIs, 2 stolen bases, and a .221 batting average. Despite a relatively disappointing start to his career (albeit with limited playing time), he broke out in the 2007 post-season, leading the Diamondbacks to a three-game sweep of the Chicago Cubs, making it to the NLCS. He finished the 2007 post-season with an impressive .357 batting average, a double, a triple, a stolen base, and an RBI to go along with an impressive .571 slugging percentage.

In 2008 spring training, Upton hit over .300 with three home runs, 12 RBIs, and four stolen bases, earning the starting right field job over Jeff Salazar. Manager Bob Melvin said that Upton has a “very high ceiling,” and that it “wouldn’t surprise him” if he had a great 2008 season.

On July 6, 2008, Upton hit a 484-foot (148 m) shot off San Diego Padres’ pitcher Josh Banks. It was at that point the second-longest home run in the history of Chase Field. The only longer home run was a 503 ft (153 m) shot off the bat of Richie Sexson.

It was in May, however where Upton turned his season around, putting up 7 home runs, 21 RBIs, 8 doubles, 4 triples, a league-leading .709 slugging percentage, to go along with a .373 batting average and a .444 on-base percentage. On June 2, 2009, Upton was named NL Player of the Month of May by the MLB for his great month. His brother Melvin Upton, Jr. earned AL Player of the Month of June, making Justin and B.J. the first pair of brothers to win player of the month honors in the same year.

As of October 10, 2009, Upton led the Diamondbacks in batting average (.300) and hits (158) to go along with 26 home runs (including his first career grand slam on 6/2/09 against the Dodgers) and 86 RBI. He’s also thirteenth in the NL in slugging percentage (.532, third among outfielders) and has compiled 20 stolen bases to go along with 7 triples and 30 doubles. On August 5, 2009 against the Pittsburgh Pirates, Upton strained his right oblique trying to steal second base, resulting in a stint on the disabled list. Rookie outfielder Trent Oeltjen had taken over his spot on the roster.

On September 1, 2009, against the Colorado Rockies, Upton was taken out in the top of the third inning after hitting a 420-foot (130 m) single in his previous at-bat. Thinking the ball to be a home run, he did not hustle to first base, and when the ball short-hopped the wall, he was forced to stay on first base.

In 2009, he led all major league outfielders in errors, with 12, and had the lowest fielding percentage among them (.961). Despite this, Justin Upton posted a 7.4 UZR, a statistic some believe to be more telling than errors and fielding%.

On March 3, 2010, Upton agreed to a six-year, $50 million extension with a $1.25 million signing bonus, as well as a limited no-trade clause.

In 2011, Upton batted .289 with 31 home runs, but on defense he led all major league outfielders in errors, with 13. He also led the NL in being hit by the pitch, with 19 (a Diamondbacks record), and was second in the league in doubles (39) and third in runs scored (105) and extra base hits (75). He won a Fielding Bible Award in 2011 as the best fielding right fielder in MLB.

Through 2011, Upton was 2nd all-time of Diamondback hitters in HBP (30), 3rd in slugging percentage (.487) and OPS (.845), 4th in OBP (.357) and strikeouts (573), and 5th in triples (24). He was awarded one of the three National League OF Silver Slugger awards.

On August 3, 2012, Justin Upton hit his 100th career home run against the Philadelphia Phillies. His brother, B.J. would hit his 100th career home run thirty minutes later in another ballgame. Upton ended the 2012 season tied for 2nd in the National League in runs scored (107; behind Ryan Braun).

After the 2012 season, Upton rejected a trade to the Seattle Mariners.

On January 20, 2013, the Arizona Diamondbacks traded Upton and Chris Johnson to the Atlanta Braves in exchange for Martín Prado, Randall Delgado, shortstop prospect Nick Ahmed, pitching prospect Zeke Spruill, and minor league first baseman Brandon Drury. The trade allowed Upton to play with his brother Melvin, who had signed with Atlanta earlier in the offseason. He and his brother hit back to back home runs on April 23, 2013 against the Colorado Rockies, marking the first time that brothers hit back to back home runs since 1938. The First time Melvin and Justin homered in the same game was April 7, 2013 when Melvin hit a game-tying home run off Carlos Marmol in the 9th inning and Justin Upton hit a game-winning home run (also off Marmol in the 9th inning) to lead the Braves a 6-5 victory over the Cubs.

On December 19, 2014, the Braves traded Upton and Aaron Northcraft to the San Diego Padres in exchange for Max Fried, Jace Peterson, Dustin Peterson, and Mallex Smith.

Upton was named co-American League Player of the Week for the week of September 19–25 (sharing the honor with Carlos Santana of the Cleveland Indians). Upton was 10-for-25 (.400) with three doubles, three home runs, six RBI, and an AL-leading 22 total bases. It was his first Player of the Week award in the American League and fourth overall, having last won the NL award on April 13, 2014 with the Atlanta Braves.

On July 6, 2015, Upton was selected as an All Star for the third time in his career, and his first selection since 2011. On October 23, 2015, Upton, along with Starling Marte and Christian Yelich, was selected as a Gold Glove finalist in LF. In his only season for the Padres, Upton hit .251 with 26 HRs and 81 RBI. He filed for free agency on November 2, 2015.

On January 20, 2016, the Detroit Tigers signed Upton to a six-year, $132.75 million contract. The contract included an option for Upton to pursue free agency again after the 2017 season. Upton hit his 200th career home run on July 18, 2016 off Minnesota Twins starter Ricky Nolasco, accounting for the only run in a 1–0 Tigers victory.

For the 2016 season, Upton hit .246 with 31 home runs and 87 RBI. 18 of Upton’s home runs and 41 of his RBI came in the last six weeks of the season, during which he was one of the hottest hitters in all of baseball.

On August 31, 2017, the Tigers traded Upton and cash to the Los Angeles Angels for Grayson Long and a player to be named later or cash. On September 15, the trade was completed with the Tigers acquiring Angels pitching prospect Elvin Rodriguez as the PTBNL.

Upton finished 2017 with a .273 batting average and .361 on-base percentage, while setting career highs with 44 doubles, 35 home runs, 109 RBI, and a .901 OPS. He also scored 100 runs for the third time in his career.

In 2019, Upton batted .215 (the lowest batting average of his career)/.309/.416 with 12 home runs and 40 RBIs. However, Upton played in just 63 games with a total of 256 plate appearances.

Upton hit his 300th career home run on July 29, 2020 in a game against the Seattle Mariners.