"The ambassador of the United States of America to the Republic of Belarus, Karen Stewart, departed the embassy at 1 p.m. ... en route to Washington," the U.S. Embassy in Minsk said in a statement.
"Ambassador Stewart's absence is temporary, and she remains the U.S. ambassador to Belarus."
Stewart's departure follows a recommendation last Friday by authorities in Belarus, accused in the West of violating fundamental rights, that she leave Minsk.
Belarus at the same time recalled its ambassador to the United States.
Officials in Washington have described Belarus, led by President Alexander Lukashenko since 1994, as Europe's last dictatorship. The country has for the past year tried to improve its ties with the West, particularly the European Union.
The embassy statement said U.S. policy toward Belarus remained unchanged and issued a new call for the country to uphold democratic principles, including the release of what Western countries call "political prisoners."
"Following the unconditional release of all political prisoners, the United States stands ready to explore steps to improve our bilateral relations," the statement said.
As part of moves to improve relations with the West, courts have in recent months freed several opposition activists considered by the West to be political prisoners.
But the country's most prominent detainee, Alexander Kozulin, is still serving a 5 1/2-year term for helping organize protests against the president's re-election.
Another detainee, Andrei Kim, is being held in connection with protests in January by small businessmen.
The U.S. State Department initially said Stewart, one of the most outspoken ambassadors in Minsk, would stay put, as no specific order telling her to go had been issued.
A prominent opposition figure in the country of 10 million, Anatoly Lebedko, said earlier that Stewart had told him she was leaving "after Belarussian authorities again recommended that she go. She sees this as a temporary departure."
Belarussian officials said they acted in response to what they saw as new sanctions against national oil-products firm Belneftekhim.
Western countries have imposed a range of sanctions against Belarus.
The United States and European Union have barred entry to Lukashenko on grounds that he rigged his 2006 re-election.
Lukashenko has sought improved relations with the West after quarrelling with Russia over energy prices. EU countries have suggested in recent weeks that a September parliamentary election could lead to better ties.
But the president threatened to expel Stewart late last year if new sanctions were imposed against his country, which is wedged between Russia and three EU countries.
Sanctions were introduced against Belneftekhim last year, prohibiting U.S. citizens from doing business with it and freezing any assets it has under U.S. jurisdiction. U.S. officials say a note issued last week implied no new punitive measures.