Isner 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.
"This transition won't be easy but I'm looking forward to every second of it with my amazing family," said Isner, who has four children with is wife Madison.
"The @usopen will be my final event. Time to lace 'em up one last time."
The towering North Carolina native crushed an ATP-record 14,411 aces in his career and has the fastest serve ever officially recorded, a 157.2 mph (253 kph) bomb he unleashed in the first round of the 2016 Davis Cup.
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. Wimbledon would later institute a rule change to ensure a similarly long match would not take place again.
The All-England Club was also the site of his best result at a Grand Slam tournament, having reached the semifinals in 2018. He twice made the US Open quarters, in 2011 and 2018.