The investment in Leeds United, which currently plays in the Championship second-tier of English football, was made by 49ers Enterprises, the NFL team's investment arm, the English club said in a statement. Financial terms weren't disclosed.
The cash infusion will go towards supplementing Leeds United's squad and mounting a more serious bid for promotion to the Premier League, which is one of the most lucrative soccer leagues in the world.
Leeds finished the 2017-2018 season in 13th place, 15 points outside the play-offs. There are 24 teams in the second division of English soccer.
The club last won the top division title in England in 1992 and hasn't played in the Premier League since 2004.
NFL owners have been attracted to English soccer given the global popularity of teams as well as the potential to pocket millions of dollars in revenue from television rights to the Premier League.
Los Angeles Rams owner Stan Kroenke is in charge at Arsenal, while Shahid Khan of the Jacksonville Jaguars has owned second-division London side Fulham since 2013. Khan is also in negotiations to buy Wembley Stadium, home of England's national soccer team.
Champions League finalists Liverpool have been under the control of John W. Henry's Fenway Sports Group, which also owns the Boston Red Sox.
However, Trans-Atlantic American investments in Premier League teams haven't always gone according to plan. Aston Villa and Sunderland were both relegated from the top division under U.S. ownership, although both clubs have since moved on to new regimes.