The US has expelled two Cuban diplomats to the UN in New York, accusing them of conducting "activities harmful to US national security", a State Department spokeswoman said.
All of Cuba's UN mission are now restricted to Manhattan where the UN is headquartered, Morgan Ortagus added.
The US has yet to provide details about the alleged actions.
Cuba's foreign minister Bruno Rodriguez called the move "categorically unjustified".
Mr Rodriguez tweeted (in Spanish) that the accusations were "vulgar slander", and said the expulsion has created tensions between the two countries.
World leaders are set to gather for an annual meeting at the UN in just a few days.
These expulsions mark the most recent diplomatic confrontation between the US and the Communist state.
In July, Cuba dismissed a US academic study which found brain abnormalities in US diplomats who worked in the country.
The US has accused Cuba of conducting "sonic attacks", after several diplomats complained of unexplained symptoms including dizziness and hearing loss.
Published in the Journal of the American Medical Association, the research suggested that the brains of the envoys had less white matter.
Academics at the Cuban Neuroscience Center said the results were unclear. A Canadian study published on Thursday suggested neurotoxins in pesticides commonly sprayed in Cuba to tackle disease-carrying insects could be the cause.
President Donald Trump has steadily increased US pressure on Cuba since taking office, changing tack from his predecessor's efforts to normalise relations with the Communist state.
The administration announced a new ban on US tour groups travelling to the Caribbean island nation in June.
Treasury Secretary Steven Mnuchin said the restrictions would "help to keep US dollars out of the hands of Cuban military, intelligence and security services".