Former ambassadors to the Holy See said moving that embassy would diminish the stature of the mission and conservative Catholic activists seized on the issue.
Addressing the growing controversy in Rome, the State Department arranged a briefing for reporters on Monday with an unnamed senior official who said the purpose for the move was to save money and increase security.
A spokesman for the Vatican said the move was well within the Holy See's requirements for embassies and that relations with the United States are far from strained.
The Rev. Thomas Rosica, a Canadian priest who works with the Vatican's press office, said the Vatican requires foreign embassies to the Holy See be separate from the country's mission to Italy, have a separate address and have a separate entrance.
Both Rosica and the senior State Department official said the proposed U.S. move satisfies those requirements.
Rosica also praised Ken Hackett, the new U.S. ambassador to the Holy See, telling CNN that "at this critical time in history, he brings eminent credentials to represent the United States to the Vatican."
He added there "a very good feeling right now" between the two countries.
Another Vatican official, not authorized to speak on the record about diplomatic relations, told CNN the Holy See understands security concerns are an issue for some countries and this move is "an exception, not the ideal, but not the end of the world."