The United States says about 20 of its military personnel will leave Ecuador by the end of April, to comply with an order from the government.
Earlier this month, President Rafael Correa told the US Department of Defense to leave amid concerns of meddling in Ecuador's internal affairs.
The order does not affect the US military attache in Ecuador.
A US spokesman in the Ecuadorian capital, Quito, said the US regretted Mr Correa's decision.
"Our close military cooperation over the past four decades has produced major advances in the fight against drug trafficking, human trafficking, terrorism and other transnational crimes," Jeffrey Weinshenker, spokesman for the US Embassy, told BBC Mundo.
He said that the US respected the "sovereign decision of the government of Ecuador", adding it could affect bilateral relations.
'Too many officers'
The expulsions make good on a months-old threat by Mr Correa to drastically reduce the number of Pentagon personnel in his country.
Last December, the left-wing president complained that the US had "a very high number" of military officers in Ecuador.
Relations between Quito and Washington have been strained ever since Mr Correa came to office in 2007.
In 2009, Ecuador refused to renew an agreement with the US that allowed its drug-interdiction flights to be based at an Ecuadorean airfield.
That same year, the Andean nation expelled two US diplomats, accusing them of meddling in its internal affairs - charges Washington rejected.
In 2012, it provided asylum in its London embassy to the WikiLeaks founder Julian Assange, whose organisation published leaked US military documents and diplomatic cables.