The move on Monday came after the US military was evicted by the Central Asian nation in a human rights dispute and ends a four-year stay at the facility.
Uzbekistan, a former Soviet republic, had permitted US forces to use the Karshi-Khanabad Air Base, called K-2, to support operations in neighbouring Afghanistan since shortly after the 11 September 2001 attacks on the US.
In July this year, President Islam Karimov gave the US six months to leave the base following the latter's criticism of the Uzbek government's suppression of demonstrators in the town of Andizhan in May.
The US pullout comes two months early.
Navy Lieutenant Commander Joe Carpenter, a Pentagon spokesman, said US personnel held a flag-lowering ceremony at the base and formally returned control to the Uzbek government.
"At 3.40pm this afternoon, a C-17 (aircraft) with 90 US personnel departed. Those were the last US personnel at the K-2 air base," Carpenter said.
The US military troop presence at the Soviet-era base had been as high as 1500. It had used cargo, refuelling and other types of aircraft at K-2, most of which have been moved to locations inside Afghanistan, Carpenter said.
Witnesses say 500 were killed
when troops fired into a crowd
The departure comes one week after the Pentagon, despite objections in the US Congress, made a payment of $22.9 million to Uzbekistan covering use of the base for the period from January 2003 to March 2005.
The Senate voted in October to delay the payment, with senators arguing the US should not pay a corrupt, repressive government that had evicted US troops.
But the measure was part of a bill that had not been finished.
Carpenter said he did not know how much the Pentagon would owe Uzbekistan for use of the base since March.
"K-2 has been extremely beneficial to combat operations in Afghanistan against the al-Qaida and Taliban and the delivery of humanitarian aid into northern Afghanistan," he said.
Carpenter added that being deprived of the use of K-2 had made operations into Afghanistan "more complicated and to some degree, more costly".
He said the base's location in Uzbekistan also afforded a land route into northern Afghanistan that alternative locations in Central Asia could not permit.
The United States still has access to an air base in Kyrgyzstan, another former Soviet Central Asian state.
Since the Andizhan killings in May, the State Department has increasingly criticised Karimov.
It recently condemned a trial that convicted 15 men of a terrorist plot over that incident.
Witnesses say about 500 people were killed when troops fired into a crowd in Andizhan on 13 May to put down a rebellion.
The Uzbek authorities put the number at 187 - mostly "foreign-paid terrorists".
Uzbekistan has also been criticised by human rights groups for jailing dissident Muslims and using torture.
Despite the eviction, the US still has permission to fly through Uzbek air space.