The American hit a backhand winner to win the last of the match's 980 points, and he took the fifth set Thursday against Nicolas Mahut, 70-68.
The first-round match took 11 hours, 5 minutes over three days, lasting so long it was suspended because of darkness - two nights in a row. Play resumed Thursday at 59-all and continued for more than an hour before Isner won 6-4, 3-6, 6-7 (7), 7-6 (3), 70-68.
The American finished with 112 aces, and Mahut had 103, with both totals eclipsing the previous high of 78. There were only three service breaks in the match, the last coming on the final point.
With Mahut serving at 68-69, Isner hit a forehand winner for the first break-point chance of the day. Then he cracked the backhand winner for the victory - only the third service break of the match, and the only one in the fifth set, which lasted 8 hours, 11 minutes.
The finish attracted an overflow crowd on cozy Court 18. Absent was Queen Elizabeth II, who had already departed the All England Club following her first visit to Wimbledon since 1977. She watched Briton Andy Murray win his match on Centre Court.
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Isner won a match that was by far the longest in the sport's history in terms of games or time. The previous longest match took 6 hours, 33 minutes at the 2004 French Open.
Four times on Wednesday, Isner was one point from victory, but Mahut saved each match point.
The crowd roared when the players walked on the court Thursday, and Isner smiled and waved. When the action resumed, both players continued to dominate with their serves. Mahut held at love five times.
But in the final game, the Frenchman committed an unforced error and netted a drop shot on consecutive points, opening the door for Isner.
Isner finished with a total of 112 aces and Muhat 103.
The first-round match surpassed the 11-hour mark stretching over three days. The fifth set alone went over 8 hours.