The first and second secretaries of the embassy have been declared persona non grata and must leave the country within seven days, the Russian foreign ministry announced on its website.
They have been accused of interfering in Russia's internal affairs, the statement said, adding that the pair had allegedly recruited a Russian citizen to obtain secret information to the detriment of Russia's security.
US ambassador Lynne Tracy was summoned to the Foreign Ministry in Moscow to receive the news.
At the end of August, Russia's FSB secret service arrested a former Russian employee of the US consulate in Vladivostok.
The authorities accuse the man of collecting information on Russia's war in Ukraine, among other things, and passing it on to the US embassy.
The suspect has been in custody since.
The incident threatens to further strain the already tense relationship between Russia and the US.
Earlier this year, Russian intelligence also arrested US correspondent Evan Gershkovich of the Wall Street Journal on espionage charges.
The accused and the newspaper deny the accusations.
Gershkovich has been in custody since the end of March.
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