The three-time grand slam champion - two of which have come at SW19 - will not add a fourth to his collection after he was downed by the big-serving American 6-4 7-6 (7-4) 6-7 (3-7) 6-4 on Centre Court.
Yet the 35-year-old did not go down quietly against the number 20 seed, forcing a tie-break in the third set to prevent a straight-sets win for his opponent before ultimately fading in the fourth.
For Murray, it marks the earliest exit of his Wimbledon career, having previously always reached the third round at senior level.
Despite never dropping more than two games behind Isner across the contest, the Scot was crucially never able to break serve.
Isner sent down a stunning 36 aces, including three in succession in the final game of the match to put him on the brink of victory, before a neat backhand over the net sealed the deal.