Murray went down 3-6 4-6 as the sixth-seeded Czech set up a semi-final against world number three Rafael Nadal or world number five Roger Federer.
Scot Murray, playing in only his second tournament since winning Wimbledon, lost in the third round of the Masters tournament in Montreal last week to Ernests Gulbis after a month’s break from the court, and reached the final of the doubles.
The British world number two has said he was keen to gain as much game time as possible this week ahead of the Grand Slam later this month at Flushing Meadows.
However he was unable to get into his groove against Berdych, despite having begun every previous match in Cincinnati on fire.
Chasing his third Cincinnati title, the first set went with serve until Berdych broke the Wimbledon champion for a 5-3 lead, with Murray furious after handing over the break point with a forehand error.
Berdych served out the set and was able to earn his second and final break in the match in the third game of the second stanza. Murray saved two break points but sent a forehand long on the third.
He was unable to break back, and the match was over when he returned a huge Berdych serve into the net.
Murray said he struggled to come to grips with the fast surface but the sixth-seeded Berdych was always going to represent a tricky test for the Wimbledon champion with the Czech holding a slight 5-4 advantage in head-to-head meetings.
The Scot, a two-time winner in Cincinnati, had just four break chances against Berdych and failed to convert them all.
"You know, the things that I really need to do well - serving, returning, moving - that was good," said Murray.
"But I'll need to work on my groundstrokes a bit, make sure I'm not making too many mistakes going into the U.S. Open.
"Today when I had my opportunities, you know, I didn't take them. That was the problem today."