The pair slugged it out on Centre Court for six hours and 35 minutes before the eighth seed came through 7-6 (8-6) 6-7 (5-7) 6-7 (9-11) 6-4 26-24.
The semi-final was deep into the fifth set when the new record was set, although it is still a long way behind the 11 hours and 5 minutes it took Isner to beat Nicolas Mahut in the 2010 first round.
Eighth-seeded Anderson's prize for his bittersweet victory over his former USA college friend and rival is a date on Sunday with either world No.1 Rafael Nadal or fellow grand slam giant Novak Djokovic.
It was also the second longest match at a Slam, beating the six hours and 33 minutes which Fabrice Santoro spent seeing off Arnaud Clement in the 2004 French Open.
Charles Oakley gets arrested for alleged cheating at a casino
He has since filed a lawsuit against Dolan and MSG alleging he was a victim of assault, battery, false imprisonment and slander. TMZ reported further details, saying that Oakley pulled back a $100 chip once he realized he was about to lose his bet.
For all the commendable fitness and focus displayed by Isner and Anderson, the match was not great entertainment.
"Especially after his win in Miami, sort of just seeing him around, watching him play matches on the practice court, I think he has a lot of confidence right now which allows him to be a bit more free from the baseline, from the back". Because of the length of the first match, that semifinal will be played with the roof closed.
For the first time in the Open era, the semi-finals were made up exclusively of players over 30.
Isner appeared to be struggling at times from 12-all to 18-all, but he held firm on his serve as Anderson, playing his second-consecutive five-set match, found a second (or third or fourth) wind. Isner in particularly started to appear worn out at the set wore on.
Anderson had break points in the 15th, 21st and 35th games of the decider before taking victory when a tired Isner hit long.