The British two-time champion, 35, struggled to tame the giant American’s serve, failing to break once in the match on Centre Court.
“I am most definitely not a better tennis player than Andy Murray. I might have been a little bit better than him today,” said Isner.
Former world number one Murray, who has slipped to 52nd in the rankings, looked to be heading out in straight sets but clawed his way back into the contest by taking the third-set tie-break, celebrating wildly in front of his passionate fans.
But, crucially, he was broken in the fifth game of the fourth set by the 20th seed, leaving him with an enormous uphill task.
Isner, 37, made no mistake, firing down another barrage of aces to come out on top for the first time in nine meetings between the pair.
The British star has a stellar record at Wimbledon, winning the trophy in 2013 and 2016.
His previous earliest exits came in the third round in 2005 and 2021.
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