The 38-year-old American reached a career-high number eight in the world rankings after a run to the Wimbledon semi-final in 2018.
John Isner will retire after the upcoming US Open, he announced on social media on Wednesday, bringing an end to a career that included a Wimbledon semi-final appearance and a victory in the longest match in tennis history.
"This transition won't be easy but I'm looking forward to every second of it with my amazing family," the big-serving, 6-foot-10 (2.08-meter) American wrote on social media.
"Time to lace 'em up one last time," the 38-year-old said, referring to the last Slam of the year, which begins in New York on Monday.
Isner reached a career-high ranking of No. 8 in 2018, shortly after reaching the semi-final at Wimbledon, where he lost to Kevin Anderson.
Isner played in the longest match in tennis history, with the American emerging the winner after winning 70-68 in the fifth set against Nicolas Mahut at Wimbledon in 2010. The match lasted just over 11 hours, across three days.
He won 16 singles titles, including a Masters 1000 title at the Miami Open in 2018, and has hit more than 14,000 aces, an ATP Tour record.
He was born in North Carolina and played tennis at the University of Georgia before turning pro in 2007.