Roughly a dozen bombers had been based at the airfield, about 160km west of London, prior to the sudden departure.
"While operational security precluded us from confirming the movement of our assets and forces in real-time, we can acknowledge now that all US bombers that were deployed to RAF Fairford have re-deployed to their home stations in the United States," a Pentagon spokesperson told the Reuters news agency.
The withdrawal comes as the US and Israel remain embroiled in a war against Iran that began on February 28. RAF Fairford has served as a stopping point for US bombers participating in the conflict, but has also been rumoured to be the target of a plot involving Iran.
On September 27, UK authorities arrested five men on suspicion of plotting to plant explosives near the base. Iran denied any such scheme, and the men were released without charge a day later. Investigators found petrol, but no explosive devices, in their vans, and UK officials say they remain under investigation.
On Thursday, British police announced the arrest of a 25-year-old UK-Iranian dual national in London on suspicion of plotting an attack against the airbase.
UK Prime Minister Andy Burnham suggested last week that Iran "played a part" in the alleged plot, though he declined to provide details.
"The investigation into circumstances surrounding events in Gloucestershire is hugely complex, and our specialist teams are interrogating multiple lines of enquiry," said Vicki Evans, Senior National Coordinator for Counter Terrorism Policing. "As we've made clear, we're looking at all possible angles, including possible foreign state involvement."
US President Donald Trump accused the men of seeking to do "big damage" to US facilities. "The arrest in the UK was fantastic. Working with Britain, it was an amazing job. We had them under investigation. We had them under view for a long time, and we got them," he said.
Iran has summoned the UK ambassador to Tehran in protest over the arrests.
RAF Fairford has hosted US forces during Middle East operations since the Gulf War in the 1990s, and has been the site of antiwar protests, including a 2003 break-in by five protesters seeking to destroy equipment bound for Iraq.