State Department spokeswoman Victoria Nuland called the 107-104 vote, with 52 abstentions, "regrettable" and "premature" and said it undermines efforts to bring Israel and Palestine back to the bargaining table for peace negotiations.
"The United States remains steadfast in its support for the establishment of an independent and sovereign Palestinian state, but such a state can only be realized through direct negotiations between the Israelis and the Palestinians," Nuland said.
She said the United States would not make a $60 million payment to UNESCO scheduled for November.
Nuland cited a legal requirement that the United States stop UNESCO funding to any U.N. agency that recognizes a Palestinian state.
Of countries that voted for Palestinian membership in UNESCO, Nuland said: "Those countries obviously made their own national decisions on this vote. We disagree with them. We made clear that we disagree with them before the vote. We made clear that we disagree with them after the vote."
Before the vote, Palestinian Authority Foreign Ministry official Omar Awadallah told the Financial Times: "It is the right of the Palestinians to become members of UNESCO.
"Palestine has its own heritage and its own culture that need to be protected," said Awadallah, who heads the authority's Foreign Ministry section dealing with U.N. bodies.
The PA, which submitted a bid for full recognition to the U.N. Security Council Sept. 24 as a path toward statehood, will also seek membership in other international bodies, including the World Health Organization and the World Trade Organization, Awadallah told the newspaper.
U.S. and European officials had appealed to Palestinian President Mahmoud Abbas to call off or postpone the vote.
The United States cannot veto Palestinian UNESCO membership, as it can motions brought to the U.N. Security Council.
"I am very much worried about the future of this organization," UNESCO Director-General Irina Bokova told the Financial Times.
UNESCO is a major global development agency whose missions include promoting literacy, science, clean water and education, including sex education and equal treatment for girls and young women.