He played in Major League Baseball (MLB) for the Pittsburgh Pirates, Boston Red Sox, Milwaukee Brewers, Washington Nationals, and Texas Rangers.

[9] The Pittsburgh Pirates selected Holt in the ninth round of the 2009 Major League Baseball Draft, one pick before his Rice teammate Ryan Berry.

[10] Holt began his professional career with the Pittsburgh Pirates' short season, Class-A affiliate, the State College Spikes, before advancing to the High-A Bradenton Marauders in 2010.

On July 13, 2011, Holt was named the MVP of the Eastern League All-Star Game after hitting a two-run homer in the ninth inning.

On June 1, while starting at first base for the first time in his career, he went 4-for-4 with four doubles, a walk, and two RBIs against the Tampa Bay Rays.

On July 9, against the Chicago White Sox, Holt drove in Daniel Nava in the bottom of the ninth inning for the first walk-off hit of his career.

He doubled, singled, and hit a home run off of Julio Teherán, and completed it with a triple in the 8th inning off of Sugar Ray Marimón.

[19] In July, Holt was chosen to represent the Red Sox in the MLB All-Star Game by Kansas City Royals manager Ned Yost.

[23] On May 15, Holt was ejected for the first time in his career by home plate umpire and crew chief Joe West for arguing balls and strikes.

[25] In the 2016 American League Division Series between the Red Sox and Indians, Holt was named the starting third baseman over Travis Shaw.

[27] On September 11, Holt hit a pinch-hit, three-run go-ahead home run off of Ryan Tepera of the Toronto Blue Jays in the seventh inning, giving Boston a lead they would hold onto for a 7–2 win, making them the first team of the year to clinch a postseason berth.

[28] Holt finished the campaign with seven home runs and 46 RBIs in 109 games while hitting .277 and playing all infield plus the corner outfield positions during the season.

On October 8, Holt became the first player in MLB history to hit for the cycle in the postseason, doing so in a 16–1 rout in Game 3 of the 2018 American League Division Series against the New York Yankees.

[2] The Red Sox went on to win the World Series over the Los Angeles Dodgers,[29] giving Holt his second career championship title.

[30] On April 30, Red Sox manager Alex Cora announced that Holt's rehabilitation had been stopped indefinitely, due to a right shoulder impingement.

[32] On July 25, Holt was ejected for the second time in his career by home plate umpire D. J. Reyburn for arguing balls and strikes against the New York Yankees.

On February 12, 2021, Holt signed a minor league contract with the Texas Rangers organization, his hometown team, that included an invitation to spring training.

[46][47] On August 14, 2024, he was announced as the new co-chair of The Jimmy Fund, serving alongside NESN personality Tom Caron.