The two countries have not had an exchange of ambassadors since July 2010.
The U.S. has asked three Venezuelan envoys to leave within 48 hours in retaliation against a similar move by the South American nation last week.
State Department Spokeswoman, Jennifer Psaki, said her agency had declared First Secretaries Ignacio Luis Cajal Avalos and Victor Manuel Pisani Azpurua; and Second Secretary Marcos Jose Garcia Figueredo in the Venezuelan embassy, “personae non gratae”.
They were given 48 hours to leave the U.S., Ms. Psaki told reporters at a regular news briefing on Tuesday.
She said that Washington remains open to diplomatic relationship with Venezuela, as President Nicolas Maduro announced on Monday his plan to nominate a new ambassador to the U.S.
“Venezuela also needs to show seriousness for us to be able to move forward; but recent actions, including expelling three of our diplomats, continue to make that difficult,” Ms. Psaki said.
Caracas accused the American envoys of meeting with university students and instigating protests and unrest in the oil-rich country where protests since February 12 had led to killing of some 12 people.
Ms. Psaki had rejected the accusations as “baseless and false”.
Washington-Caracas ties have not improved since Maduro took over the presidency from late Hugo Chavez in March 2013, and Maduro had expelled American diplomats twice, sparking U.S. tit-for-tat moves.
The two countries have not had an exchange of ambassadors since July 2010, though Venezuela is flowing 900,000 barrels of oil per day to the U.S.