The third-seeded Swiss held serve throughout but lost two tiebreaks against his American opponent, who will face Spain’s David Ferrer in the quarter-finals.
Federer, who won the Basel title last Sunday, seemed to be cruising after breezing through the second set, but Isner pushed him to another tiebreak, using his devastating serve to prevail in the deciders.
Isner, who fired down 27 aces, ended the contest on his fourth match point with a service winner.
“It’s tough to get out of the tournament not having lost your serve but that’s how it can go against John,” the former world number one told reporters.
“At the very end of the season, going two tournaments in a row after playing a lot of tennis in Basel the week before… there’s maybe a bit of a letdown after Basel.”
Federer’s defeat was the first upset at Bercy after world number one Novak Djokovic and second seed Andy Murray were joined by Rafa Nadal in the quarter-finals.
Nadal saved a match point before subduing South African Kevin Anderson 4-6 7-6(6) 6-2.
The seventh seeded Spaniard appeared to be on his way out but a risky forehand winner allowed him to save match point at 5-6 down in the second set tiebreak.
Anderson, who served 20 aces, cracked in the final set as Nadal secured a meeting with fourth seed Stan Wawrinka after the Swiss eased past Serbian Viktor Troicki 6-4 7-5.
Djokovic’s serve suffered a second-set malfunction against Gilles Simon but the Serb stayed on course for a third consecutive title on the banks of the River Seine with a 6-3 7-5 victory.
He dropped serve four times in the second set but Simon could not capitalise as Djokovic set up a quarter-final against Tomas Berdych, who defeated local favourite Jo-Wilfried Tsonga 6-3 6-4.
Djokovic, however, will need to raise his game if he is to win a fourth title here as his possible final opponent, Murray, looked in ominous form.
The Scot put a marker down for the Davis Cup final when he dismantled Belgium’s David Goffin 6-1 6-0 in awe-inspiring fashion to advance into the last eight.
Three weeks before Britain and Belgium face off in the Davis Cup final on clay in Ghent, Murray had just too much pace for the 16th seeded Goffin on the Bercy hard court.
Goffin can only hope the red dust will slow down world number three Murray in the Nov. 27-29 tie after the Scot had the Belgian chasing from corner to corner throughout.
An unforced error gave Murray the opening set and the Olympic champion was relentless in the second, closing it out with a backhand winner down the line having allowed Goffin only eight points on his service games.
Japanese sixth seed Kei Nishikori retired with an abdominal injury in his third-round match against Frenchman Richard Gasquet, a scare with the ATP World Tour finals 10 days away.
Nishikori was trailing 7-6(3) 4-1 when he threw in the towel against Gasquet.
The Japanese is one of eight players qualified for the World Tour finals, which will be played at the O2 Arena in London from Nov. 15-22. Djokovic, Federer, Murray, Stan Wawrinka, Rafa Nadal, Berdych and Ferrer are the others.
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