The Canadian and partner Yifan Xu of China, seeded sixth, lost to No. 12 seeds Nicole Melichar of the US, and Kveta Peschke of the Czech Republic 6-3, 4-6, 7-5 in the Wimbledon semifinals on Friday.
He then converted his first match point when Isner sent a shot wide.
John Isner laid his aces on the table but found a way into the Wimbledon semi-finals by breaking the Milos Raonic serve.
The second semifinal of the day, pitting Rafael Nadal against Novak Djokovic, was easily the most highly-anticipated match of the tournament so far - and its start was delayed well over three hours as the city of London waited for either Isner or Anderson to blink.
Anderson became the first South African male to reach the final since 1921 with a 7-6 (6), 6-7 (5), 6-7 (9), 6-4, 26-24 victory on Centre Court.
Isner has won eight of the 11 professional matches against Anderson and is projected by many to advance from this battle of two of the game's biggest servers.
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However, Isner was then unable to convert a set point of his own in the tiebreak before 32-year-old Anderson wrapped it up thanks to a netted return.
That inconsistency was eliminated in an absorbing final set before Anderson produced a moment of magic.
He finally broke the 33-year-old American in the 49th game of the decider after coming close in a couple of earlier games and looked impassive as he held his nerve to secure victory.
Isner is best known to many fans, particularly in Great Britain, for his astonishing contest with Nicolas Mahut on Court 18 at Wimbledon in 2010. The two went back and forth all the way to 12-all, essentially playing a full sixth and seventh set.
Anderson had break points in the 15th, 21st and 35th games of the decider before taking victory when a tired Isner hit long. We played each other in college probably three, four, five times.