De Minaur had the chance to climb into the world's top five with a deep run at the final Masters 1000 event before the season's last grand slam in New York.
But the Australian No.1 delivered a flat performance to fall 6-3 6-4 to rising Frenchman Arthur Fils in just 82 minutes on Wednesday (Thursday AEST).
De Minaur dropped serve once in each set and was unable to break the 21st seed at all.
The world No.8's exit means de Minaur is certain to be seeded in the top eight at Flushing Meadows, still ensuring the Australian doesn't run into a higher-ranked player until at least the quarter-finals.
But with Carlos Alcaraz still injured, de Minaur could have snatched a US Open top-four seeding and been guaranteed to avoid heavyweights such as Jannik Sinner and Alexander Zverev until at least the semi-finals.
Australian qualifier Chris O'Connell plays his fourth-round match in Cincinnati later on Thursday against American sixth seed Taylor Fritz.
In another upset, American Tommy Paul saved a match point to defeat top seed Zverev 4-6 7-6 (8-6) 6-4 and reach the quarter-finals after a nearly 90-minute rain delay.
German world No.3 Zverev took the first set and trailed 3-4 in the second before the fourth-round match was suspended due to rain.
Paul emerged from the lengthy delay looking the sharper player, saving a match point before levelling the contest as Zverev struggled to find his rhythm.
The German took a 4-2 lead in the third set but Paul dug in to take control of the match, rallying to win four straight games to advance to the quarter-finals in Cincinnati for the first time.
Paul next plays Flavio Cobolli after the Italian earlier defeated Spain's Rafael Jodar 4-6 7-6 (7-3) 6-3.
- with Reuters
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