In a lengthy written statement, Irina Bokova, Unesco's director-general, expressed regret at the American withdrawal and said that the American people shared the organization's goals.
The agency is known for designating world heritage sites such as Syria's Palmyra and the US Grand Canyon. The decision "reflects USA concerns with mounting arrears" at the United Nations entity, as well as what she called "continuing anti-Israel bias".
The agency is in the process of choosing a new leader, with Qatari and French former ministers Hamad bin Abdulaziz al-Kawari and Audrey Azoulay neck-and-neck in the contest to replace Ms Bokova.
"Despite the withholding of funding, since 2011, we have deepened the partnership between the United States and UNESCO, which has never been so meaningful", she underscored.
"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", Bokova said.More news: Now, post Instagram 'stories' directly on Facebook
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The US has long used UNESCO as a stage for political gestures: In 1984, the Ronald Reagan withdrew the US from UNESCO, accusing it of being pro-Soviet, anti-Israel, and anti-free market.
"This is another example of the Trump's administration's profound ambivalence and concern about the way the U.N.is structured and behaves, and it shows the administration's determination to separate itself from its predecessors", said Aaron David Miller, a former Middle East negotiator and adviser in Republican and Democratic administrations. Israel has accused UNESCO of making a politically motivated move, part of what it says is an attempt to deny the Jewish character and heritage of certain key sites in the Holy Land.
The withdrawal, effective December 31, 2018, won't be the first for the United States.
UNESCO's executive board plans to select her successor Friday in a secret ballot. It extended membership to Palestine in 2011. "Ideally, it steers it in way that USA interests and UNESCO's mandate will converge".
Mr Netanyahu condemned the agency this year for declaring the old city of Hebron in the West Bank a Palestinian World Heritage site.
The agency also works to improve education for girls in desperately poor countries and in scientific fields, to promote better understanding of the horrors of the Holocaust and to defend media freedom, among other activities.