Interleague play in Major League Baseball refers to regular-season baseball games played between an American League (AL) team and a National League (NL) team. Interleague play was first introduced during the 1997 Major League Baseball season. Before that, matchups between AL teams and NL teams occurred only during spring training, the All-Star Game, other exhibition games (such as the now defunct Hall of Fame Game in Cooperstown, New York), and World Series. Unlike modern interleague play, none of these contests, except for the World Series, counted toward official team or league records.

From 1997 to 2001, regular season interleague play occurred only on a geographical basis, with divisions in each league (the West, Central, and East) assigned to play their counterpart in the opposite league. In 2002, MLB introduced a system of rotating matchups, allowing interleague, cross-country games to occur in the regular season for the first time. In 2023, MLB began scheduling all 30 teams to play each other every year.

Regular season interleague play was discussed for baseball's major leagues as early as 1903, when the two major leagues made peace and formed the National Commission as governing body. The first National Commission Chairman, Cincinnati Reds president August Herrmann (who had already been a proponent of interleague play), proposed an ambitious scheme in late 1904. Herrmann's plan would have seen the two leagues ending their seasons earlier, after approximately 116 games, "and then have every National League team play two games in every American League city, and have every American League team play two games in every National League city." Another interleague play idea was floated around the same time by Boston Americans owner John Taylor, whose plan was for each league to play its full 154-game schedule, to be followed by not just a championship series between the two league winners, but also by series between the two second-place finishers, the two third-place teams, and all other corresponding finishers.

In August 1933, several owners reacted favorably to a proposal by Chicago Cubs president William Veeck to have teams play four interleague games in the middle of the season, beginning in 1934. In December 1956, Major League owners considered a proposal by Cleveland Indians general manager and minority-owner Hank Greenberg to implement limited interleague play beginning in 1958. Under Greenberg's proposal, each team would continue to play a 154-game season, with 126 within that team's league, and 28 against the eight clubs in the other league. The interleague games would be played immediately following the All-Star Game. Notably, under Greenberg's proposal, all results would count in regular season game standings and league statistics. While this proposal was not adopted, the current system shares many elements. Bill Veeck predicted in 1963 that Major League Baseball would someday have interleague play, and in 1968 included it in a proposal for a dramatic realignment of the major league structure. While the concept was again considered in the 1970s, it was not formally approved until 1996, at least in part as an effort to renew the public's interest in MLB following the 1994 players' strike.

MLB's first regular-season interleague game took place on June 12, 1997, as the Texas Rangers hosted the San Francisco Giants at The Ballpark in Arlington. There were four interleague games on the schedule that night, but the other three were played on the West Coast, so the Giants–Rangers matchup started a few hours earlier than the others. Texas's Darren Oliver threw the game's first pitch and San Francisco outfielder Glenallen Hill was the first designated hitter used in a regular-season game by a National League team. San Francisco's Darryl Hamilton got the first base hit in interleague play, while Stan Javier hit the first home run, leading the Giants to a 4–3 victory over the Rangers.

From 1997 to 2001, teams played against the same division from the other league; for example, the American League West played teams from the National League West, typically scheduled to alternate between home and away in consecutive years. In 2002, however, the league began alternating which divisions played which divisions, and thus in 2002 the American League East played the National League West, the American League Central played the National League East, and the American League West played the National League Central. Matchups which had been of particular interest prior to this format — mainly geographic rivals — were preserved. Corresponding divisions were skipped once when this rotation began, but were put back in the rotation in 2006.

From 2002 to 2012, all interleague games were played prior to the All-Star Game (with the exception of games postponed by weather that were made up after the All-Star Game). Most games were played in June and early July, although beginning in 2005, interleague games were played during one weekend in mid-May.

The designated hitter (DH) rule was originally applied in the same manner as in the World Series (and the All-Star Game prior to the 2010 edition). In an American League ballpark, both teams had the option to use a DH, while in a National League ballpark, both teams' pitchers were required to bat. Teams from both leagues have both benefited and have been at a disadvantage when it comes to the DH rule in interleague play. For instance, Barry Bonds, who spent his entire career in the National League and actually won eight Gold Gloves earlier in his career, was used strictly as a DH later in his career when the San Francisco Giants played away interleague games due to his poor fielding. Conversely, Boston Red Sox slugger David Ortiz, who spent his entire career in the American League and was the Red Sox's regular DH, was assigned to play first base when the Red Sox had away interleague games, with the Sox choosing to give up good fielding in favor of retaining Ortiz's power hitting. With the introduction of the DH to the National League in 2022, this no longer applies.