The US has expelled 15 Cuban diplomats, saying Havana failed to protect US diplomats from mysterious acoustic attacks.
Cuba’s Foreign Minister Bruno Rodriguez called the move “unacceptable”.
The Department of State move follows last week’s US withdrawal of more than half of its own diplomats from the Cuban capital.
Nearly two dozen US personnel have suffered unexplained ill health in the city.
“The decision was made due to Cuba’s failure to take appropriate steps to protect our diplomats in accordance with its obligations under the Vienna Convention.
This order will ensure equity in our respective diplomatic operations,” US Secretary of State Rex Tillerson said in a statement on Tuesday.
The Cuban diplomats have been given seven days to leave.
Mr Rodriguez said the move was “unjustified”.
At least 21 people working at the US’s embassy in Cuba have reported health problems, ranging from mild brain trauma and deafness to dizziness and nausea.
Earlier reports suggested sonic attacks were to blame, but nothing has been proven.
Cuba denies targeting embassy staff, and the US has not blamed the country’s government for the suspected attacks.