The former world No. 8 announced he would be retiring after the tournament with a social media post last week.
After a match that lasted just under four hours, 38-year-old Isner addressed the packed crowd at Grandstand through tears and sniffles.
"This is why I've worked as hard as I have my whole life, to play in atmospheres like this," Isner said. "I might not win them all, as we know, just like today. To play in front of this crowd and have the support I've had is pretty special."
As he walked off the court, he received a standing ovation from those in the stands and stopped to wave in appreciation before reaching the exit.
Isner leaves tennis having reached some of the sport's highest levels. A star at the University of Georgia, winning the NCAA doubles title in 2005 and the team title in 2007, Isner turned pro in 2007 and surprised even himself with what he was able to achieve throughout his career.
"I think I've overachieved," Isner said Friday. "I never imagined myself having this much success for this long."
Those achievements include 16 ATP singles titles, eight doubles titles, a Wimbledon semifinal appearance in 2018 and a streak of 10 seasons in which he finished in the year-end top 20. For eight of those seasons, he was the top-ranked American man. He also is the ATP's record holder for most career aces with 14,470 -- he added 48 more to his total in his final match -- and for the fastest serve at 157.2 miles per hour.
"I believe he has the best serve in the history of the game," three-time Grand Slam champion Andy Murray said Tuesday. "Amazing technique. First and second serves, could do everything with it."
In addition to his towering 6-foot-10 frame, Isner is perhaps best known for his role in the longest match in professional tennis history. During the first round at Wimbledon in 2010, Isner defeated Nicolas Mahut in a match that lasted over 11 hours, spanned three days and needed 183 games.
Currently ranked No. 157, Isner has struggled this season. His first-round win over Facundo Diaz Acosta on Tuesday marked his first major victory of the season and just his ninth win on the year. He was candid when speaking to the media about the impact his inability to play and train to the standard he would like had on his decision to retire.
"I did want to play as long as I could," Isner said. "If my results were better this year, I probably wouldn't be speaking to you right now. That just hasn't been the case.
"This year has been tough healthwise; I had a foot thing that just bothered me for a long time. I wasn't able to train really at all, but I was still playing without much practice. It just wasn't much fun. It just became very laborious for me just trying to get myself healthy and not really being able to do it. Competing not quite at 100% for a little bit too long this year, [it was] very taxing mentally on me. Then you take a few losses here and there, and it just becomes tough, a little bit discouraging."
During his two matches in New York, he had tape on his left knee and his right calf. But Isner insisted he has no regrets about his decision and is looking forward to spending time with his wife and four children, as well as pursuing other interests. He said he would likely do some television work as an analyst and explore business opportunities.
After his first-round victory Tuesday, he was honored with a ceremony at Louis Armstrong Stadium. There was a video showing some of his career highlights; he was presented with a framed picture by the tournament; and his family and friends, including former players Sam Querrey and Bob and Mike Bryan, joined him on the court.
But he was emphatic when he told the crowd he wasn't done just yet.
"It's not goodbye yet," he said. "I'm still alive. I'm actually pretty good, so I'm going to try to keep this thing going as long as I possibly can."
A few hours later, Isner headed into full-fledged retirement by dropping a doubles match alongside Jack Sock, another American who has said the US Open will be his last tournament. They were beaten by Robert Galloway and Albano Olivetti 6-2, 3-6, 7-6 (10-3) at Court 12.
Information from The Associated Press was used in this report.