Isner, 38, turned pro in 2007 and reached a career high of world number eight.
He won 16 singles titles, the biggest being the Miami Open crown in 2018, his most successful season.
"After 17+ years on the @atptour, it's time to say goodbye to professional tennis," Isner said on social media site X, formerly known as Twitter.
He came out on the winning side of the longest pro tennis match in history, which lasted 11 hours and 5 minutes and took place over three days at Wimbledon in 2010.
After 183 games in that match, Isner defeated qualifier Nicolas Mahut in the first round.
Persons:
John Isner, Taylor Fritz, Henry Romero, Isner, Madison, Nicolas Mahut, Rory Carroll, Sonali Paul
Organizations:
U.S, REUTERS, ATP, Miami, Wimbledon, Isner, England, Thomson
Locations:
Acapulco, Mexico, North Carolina, Los Angeles