While the US stopped funding Unesco after it voted to include Palestine as a member in 2011, the State Department has maintained a Unesco office at its Paris headquarters and sought to weigh in on policy behind the scenes.
Unesco director-general Irina Bokova, who is Bulgarian, expressed "profound regret" at the decision and said the departure was a loss for "the United Nations family" and for multilateralism.
She said the US and Unesco matter to each other more than ever now because "the rise of violent extremism and terrorism calls for new long-term responses for peace and security".
Ms Bokova defended Unesco's reputation, noting its efforts to support Holocaust education and train teachers to fight anti-Semitism.
She traced the decades-long US ties with Unesco, and noted that the Statue of Liberty is among the many World Heritage sites protected by the UN agency.
Ms Bokova's two terms as director have been deeply scarred by the 2011 Unesco vote to include Palestine as a member, funding troubles and repeated resolutions seen as anti-Israel.
Many saw the vote to include Palestine as evidence of long-running, ingrained anti-Israel bias within the United Nations, where Israel and its allies are far outnumbered by Arab countries and their supporters.
Unesco is best known for its World Heritage programme to protect cultural sites and traditions around the world.
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.
The Trump administration has been preparing for a likely withdrawal for months, and a decision was expected before the end of the year, according to US officials.