Japanese star Nishikori clinched a dramatic 1-6, 6-4, 4-6, 6-1, 7-5 comeback triumph in a shade under four hours against the Wimbledon and Olympics winner and 2012 US Open champion.
In Friday’s semi-final, the 26-year-old will take on Swiss third seed Stan Wawrinka who defeated tearful Juan Martin del Potro, the 2009 champion, 7-6 (7/5), 4-6, 6-3, 6-2.
Wawrinka, who had to save a match point in his third round clash with Dan Evans, will be playing in a third US Open semi-final in four years.
The other semi-final on Friday will see defending champion Novak Djokovic take on 10th seeded Frenchman Gael Monfils.
“It was one of my toughest matches mentally and physically. Juan Martin’s an incredible player,” said Wawrinka.
In a match which featured 17 breaks of serve, Nishikori prevailed for only his second win in nine matches against world number two Murray.
His win came just three weeks after losing to Murray in the Olympic semi-finals.
“It was a really difficult match. I didn’t start well. I felt it was really quick and I was missing too much,” said Nishikori after reaching only his second Slam semi-final after his runner-up spot in New York in 2014.
“In the fourth and fifth sets I think I played some of the best tennis.”
Murray looked to be in control at two sets to one ahead and carving out a break point in the third game of the fourth when a loud gong-like sound burst from the malfunctioning public address system in Arthur Ashe stadium.
Umpire Marija Cicak ordered the point to be replayed much to the irritation of the Briton who lost his composure and the next five games as an acrobatic Nishikori levelled the tie.
Murray refused to blame his defeat on the incident.
“Definitely I would say to 4-1 I didn’t play a good game after I got out of the change of ends, and then he held pretty comfortably the next game,” said Murray.
“But after that, I don’t think so. There was a lot of time.”
Nishikori broke for 1-0 in the deciding set and backed it up for 2-0 before Murray stopped a seven-game losing streak to hold.
Murray, with his focus suddenly rebooted, broke back for 2-2. But back came Nishikori, stunning his rival to break again for 3-2 before holding for 4-2 as both men thrilled with a series of big hits, subtle touches and exhausting athleticism.
Nishikori surrendered the advantage yet again, giving up a 40-0 lead in the eighth game and missing an easy volley.
The Briton was soon 5-4 up but Nishikori, showing nerves of steel, held and broke again before taking victory when Murray blinked first in the 12th game, burying a backhand in the net.
Del Potro, the world number 142 and bidding to become the lowest ranked man to reach a Grand Slam semi-final in 16 years, was out to a 4-1 lead in the first set against Wawrinka.
The Swiss broke back in the seventh game and although Del Potro, whose career was almost derailed by a series of wrist injuries, saved two set points in the tiebreaker, a rare forehand error gave up the set.
The Argentine’s bruising recent schedule had seen him claim an Olympic silver medal last month and he used that experience to claim the only break for a 4-3 lead in the second set.
But the Swiss was looking the fresher as the clock ticked towards and then past 1 a.m. He took the third set 6-3, hitting twice as many muscular winners.
Del Potro was a spent force and gave up a double break in the fourth set on his way to defeat but leaving him at least with the compensation of being back in the top 65 after being ranked as low as 1,045 in February.
He was in tears on the eve of the final game before the two men embraced at the net.
“I can lose the match but I will never forget this. It’s bigger than winning any match,” said del Potro of the ovation he received.
“I’m so proud to get that from the crowd because I’ve been doing a big effort to play tennis again.”