World number one Djokovic, the 2011 champion, reached his eighth consecutive US Open semi-final by downing Murray 7-6 (7/1), 6-7 (1/7), 6-2, 6-4 for his 13th win in 21 meetings against his childhood rival who had taken the 2012 title.
Nishikori, the 10th seed, had earlier needed more than four hours to defeat third seed Stan Wawrinka 3-6, 7-5, 7-6 (9/7), 6-7 (5/7), 6-4 to become the first Japanese man in the semi-finals in 96 years.
Wimbledon champion Djokovic and Murray had spent a record-equalling 4 hours 54 minutes contesting the 2012 final and their quarter-final clash threatened to surpass that mark when they needed over two hours to get through the first two sets of a tie that started on Wednesday and finished Thursday.
By the end, however, Murray was struggling with what appeared to be a recurrence of his long-standing back problem, a legacy of having spent three and a half hours more on court than Djokovic in getting to this stage.
But victory was also a testament to the top seed's intimidating defensive skills as he saved 12 of 16 break points in the 3-hour 32-minute encounter which saw Murray commit 65 unforced errors to Djokovic's 48.
"We both gave our best and at times the tennis was not nice but that was due to the intense physical battle in the first two sets," said Djokovic.
"I didn't expect anything less from Andy. Our last few matches have been three and four hours and he has a lot of quality on all sorts of surfaces,
"I knew it would be a tough match and that the more aggressive player would win."
Murray, still without a title since his historic 2013 Wimbledon triumph, said that he struggled as the match wore on.
"I tried my best to hang in there in the fourth set but he was fresher than me or at least he appeared to be," said Murray, who admitted that he felt stiffness in his hips and back as the night got cooler.
"The pace of my serve slowed significantly and he was a little more solid. I fought hard but it wasn't enough."
Djokovic and Nishikori are 1-1 in career meetings but their last clash was three years ago.
"It's great for Kei and Japan for him to be in the semi-final. He serves and moves very well," said the world number one who has made the final in New York in the last four years.
Djokovic raced into a 4-1 lead but Murray pulled it back to 4-4 while the ninth game featured a 29-shot rally and the 10th a 104mph forehand off the Murray racquet.
But it was the world number one who played the more composed tiebreak, helped by a third Murray double fault and a rare loose backhand volley as Djokovic nosed in front after 73 minutes.
Djokovic was 3-1 up in the second set before Murray clawed his way back to 3-3. They exchanged breaks again to 4-4 with the Serb picking up a time violation before saving a set point in the 11th game.
Murray, however, swept through the tiebreak to level the tie as the clock nudged midnight and Djokovic dropped his first set of the tournament.
For the third set in succession, Djokovic grabbed the early break at 3-1 and this time held on, even piling on the agony with another break of a suddenly lacklustre Murray to claim the set 6-2.
Clearly struggling physically, Murray cracked in the 10th game of the fourth set with the match over when he netted a backhand.
Nishikori, 24, triumphed over Australian Open champion Wawrinka in 4 hours 15 minutes, his second marathon clash after needing 4 hours 19 minutes to get past Milos Raonic in the previous round.
Ichiya Kumagae was the last Japanese man to reach the semi-finals of the US Championships in 1918.
Nishikori, whose match against Raonic ended at a record-equalling 2:26am on Tuesday, was stunned by his win over the third seeded Wawrinka, who had won the pair's two other meetings without dropping a set.
"I was tight at the start but my body was OK. I felt more confident after the first set," said the 24-year-old, who will be playing in his first Grand Slam semi-final having made the quarter-finals in Australia in 2012.
"It feels amazing. I am playing well and I hope to play at 100 percent in the semi-finals."
Wawrinka praised the stamina of Nishikori who has now spent more than 13.5 hours on court at the tournament.
"From outside he looks really dead, but we know on the court he can play," he said.