The Swiss top seed was beaten 6-4 3-6 6-1 6-4 to give 24-year-old Berdych the biggest victory of his career.
Federer had not lost before the final at SW19 since 2002 and was bidding to win a record-equalling seventh title.
But Berdych gave a performance of the very highest order to set up a semi-final meeting with Novak Djokovic.
"It's really tough to explain how I'm feeling, it's unbelievable," Berdych told BBC Sport after leaving Centre Court to a standing ovation.
"To play on this stadium against a player as great as Roger and to be standing here as the winner is amazing. This was the toughest match of my career to close out, it's a big step forward and I'm so happy.
"Right now it's really tough to think about my next match - I just want a few minutes and hours to enjoy this. Then I have to get ready and prepare for my next opponent."
Berdych becomes the first Czech man to reach the last four of Wimbledon since Ivan Lendl in 1990 and he fully deserves to be there following this historic triumph.
Having beaten Andy Murray en route to his first Grand Slam semi-final at this year's French Open and now overcome arguably the greatest tennis player of all time, the 6ft 5ins right-hander is building quite some reputation.
The result will see Federer drop to number three in the world rankings for the first time since November 2003 and he must now regroup ahead of the US Open.
"I'm definitely struggling at the moment," said the 29-year-old current world number two, who also fell in the quarter-finals at the French Open. "I'm looking forward to a rest and then I'll attack again in North America.
"Reaching the quarter-finals is a decent result. Obviously some people think it's shocking, but many players would die to play a Grand Slam quarter-final.
"I don't think I played poorly but he went after it. I was not able to defend well enough and he played well when he had to - it was brutal for me. Every time he had a chance, he took it."
Federer went on to reveal that he was suffering from back and thigh problems picked up while contesting the pre-Wimbledon grass-court event in Halle, Germany.
"I'm unhappy with the way I'm playing, I couldn't play the way I wanted to," added the Swiss, who needed five sets to overcome Alejandro Fall in the first round and four to oust Ilija Bozoljac in the second. "I'm struggling with a little bit of a back and a leg issue. They don't allow me to play the way I would like to.
"The leg issue came in the final of Halle and returned a little bit after my first-round match here. It went away again but just kept creeping back during the matches.
"For the last fix or six days the back has been really badly. It's normal that the back tends to get stiff in the grass-court season but it's just not nice when it doesn't go away and you can't play freely. That's what I was missing today."
Federer came into the match as the firm favourite but, from the moment he was taken to deuce in game one, it was clear the 16-time Grand Slam champion was not going to have everything his own way.
Berdych combined much-improved movement with jaw-dropping forehands and lightning-quick deliveries to wow the Centre Court, and broke in game seven before serving out the opening set.
There would, however, be an immediate shift in momentum as Berdych tensed up in game two of the second set and allowed Federer to break to 15 with a fine crosscourt forehand pass.
Despite squandering two more break points in games six and eight, the Basel-born right-hander levelled the match and at that point Federer would have been expected by many to run away with it.
But Berdych had other ideas, targeting Federer's unusually brittle second serve to break in games two before serving out the 27-minute third set.
Although Federer had won eight of his previous 10 meetings with Berdych, the Swiss was reduced to tears after losing their first encounter at the Athens Olympics and also succumbed in their most recent duel at April's Miami Masters.
So he knew all about the world number 13's threat, yet seemed powerless to contain it.
Federer looked set to make the first move in a pulsating fourth set but Berdych dug deep to escape from 0-40 in game six and then forced the decisive break when Federer volleyed wide in game seven.
Berdych was soon serving for the match and the pressure appeared to have got the better of him as Federer saved a match point and brought up a break-back point.
But the Czech held his nerve to unleash his 51st and final winner and confirm the biggest upset of the 2010 Championships.
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