The team of Tianna Madison, Allyson Felix, Bianca Knight and Carmelita Jeter sprinted to an emphatic win in 40.82, shattering the East German mark of 41.37 which had stood for 27 years.
The Jamaicans ran a national record of 41.41, but still finished well adrift in second while Ukraine took the bronze.
It was a measure of revenge for the USA, who have seen Jamaica once again dominate the sprints at London 2012, last night completing a clean sweep in the men's 200m.
Jeter said: 'I knew we had won. Everyone ran an incredible leg. We were able to get the stick around and we were able to get the gold medal and the world record, I knew that right away.
'I knew we were moving, I knew we were running very well and so I was excited.'
Felix, who won the 200m, said: 'It is a relief, it is a joy, it is everything.
I knew we had won. Everyone ran an incredible leg.Carmelita Jeter
'We went into this race the most comfortable I have seen this team. We were laughing and smiling. We have never been like that.'
France's Renaud Lavillenie took gold in a thrilling Pole Vault final in an Olympic record 5.97m.
Lavillenie looked set for bronze when he failed his first attempt at 5.91m, but passed the height before making his second and final attempt at the next one.
Germany's Bjorn Otto was second with 5.91m, ahead of fellow countryman Raphael Holzdeppe on countback.
'I can't really believe it,' the Frenchman said. 'Maybe tomorrow I will wake up and realise that I am an Olympic champion.
'The Germans were very strong and they pushed me to my limits. This has to be the best thing in my life.'
The quartet of Chris Brown, Demetrius Pinder, Michael Mathieu and Ramon Miller ran a national record 2:56.72 to finish 0.33 ahead of the USA, with Trinidad taking the bronze.
The USA team were watched by Manteo Mitchell, who ran on a broken leg in yesterday's semi-final.
'I am heartbroken for them. You never know what I could have done,' he said.
The race also saw Oscar Pistorius bringing the South African team home in eighth place.
He said: 'It has been a great achievement for me to come here and compete. I never set out to prove a point, but I do feel I have made the point. It's been a privilege to be on the track with all the other phenomenal athletes.'
In the women's 1500m, Asli Cakir Alptekin of Turkey took gold, winning a slow race in 4:10.23.
Alptekin finished ahead of compatriot Gamze Bulut, with Bahrain's Maryam Yusuf Jamal third.
There was more agony for American Morgan Uceny as she fell for the second major final in succession, having also hit the track at last year's World Championships in Daegu when she was one of the favourites.
Russia's Tatyana Lysenko won gold in the women's Hammer with an Olympic record of 78.18m.
She led all the way through the competition, having thrown 77.56m with her first attempt, before going even further with her penultimate effort.
Poland's Anita Wlodarczyk took the silver with a final throw of 77.60m, while the bronze went to Germany's Betty Heidler.
Ethiopia's Meseret Defar claimed gold in the 5000m final, denying her compatriot Tirunesh Dibaba the chance to add another title to the 10,000m crown she won a week ago.
Defar, who won 5000m gold eight years ago at Athens 2004, held off her challengers down the home straight to win in 15:04.25.
Kenya's Vivian Cheruiyot was second with Dibaba getting the bronze.
Defar said: 'I'm very happy. It's a great day for me. Since 2008 I have tried everything as I wasn't able to win the Olympics.'
In the semi-finals of the men's 4 x 100m, the USA ran a national record of 37.38 to qualify faster than Jamaica, though they still have Usain Bolt to bring in.