The head of Unesco, Irina Bokova, voiced "profound regret" over the decision, calling it a "loss to multilateralism".
"At the time when conflicts continue to tear apart societies across the world, it is deeply regrettable for the United States to withdraw from the United Nations agency promoting education for peace and protecting culture under attack," she said.
In a statement, Bokova said that "despite the withholding of funding, since 2011, we have deepened the partnership between the United States and Unesco, which has never been so meaningful."
UNESCO, which is best known for producing the list of World Heritage sites that includes the Grand Canyon and other US attractions, has been the scene of diplomatic flare-ups in recent years after Arab countries succeeded in passing a number of resolutions critical of Israel.
In May this year, Israel was infuriated by a resolution identifying Israel as "the occupying power" in the divided of city of Jerusalem and calling on it to rescind any move changing the city's "character and status".
The text denounced "all legislative and administrative measures and actions taken by Israel, the occupying power, which have altered or purport to alter the character and status of the Holy City of Jerusalem."
In July, the agency again delighted Palestinians when it declared the Old City of Hebron in the occupied West Bank an endangered World Heritage site.
Israeli Prime Minister Benjamin Netanyahu called the vote "another delusional decision" by Unesco, which promotes education, cultural development and free media initiatives around the world.
The agency's board members are set to vote this week to elect a new director-general, with candidates from Qatar, France and Egypt in a three-way race for the position.