The Cuban personnel have been given seven days to leave.
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 ill health after unexplained attacks in the Cuban capital.
"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.
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.
What is a covert sonic weapon?
US-Cuba thaw halted amid diplomat injuries
Cuba denies targeting embassy staff, and the US has not blamed the country's government for the suspected attacks.
"We continue to maintain diplomatic relations with Cuba, and will continue to co-operate with Cuba as we pursue the investigation into these attacks," Mr Tillerson added in his latest statement.
Last week US officials said: "We don't know the means, the methods or how the attacks are being carried out."
Cuba mystery attacks
Late 2016: US embassy staff and at least one Canadian began to notice symptoms
May 2017: US expels two Cuban diplomats for failing to protect its diplomats
August: US says 16 employees have been treated but attacks seem to have stopped
Early September: US says attacks are continuing and 19 staff members have now been hurt
29 September: Washington pulls diplomatic staff, warns US citizens not to visit and says 21 embassy employees now injured
3 October: US expels Cuban diplomats from Washington
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