The US decision, announced in Washington, follows years of tension at the organisation which it accused of having an "anti-Israel bias".
"This decision was not taken lightly, and reflects US concerns with mounting arrears at UNESCO, the need for fundamental reform in the organisation, and continuing anti-Israel bias at UNESCO," US State Department spokeswoman Heather Nauert said.
The United States has walked out of the 195-member organisation once before under ex-president Ronald Reagan, who quit in 1984 over alleged financial mismanagement and claims of anti-US bias in some of its policies.
President George W. Bush announced America's return in 2002, but relations soured again in 2011 when Barack Obama pulled the plug on funding to the body after its members voted to admit Palestine as a full member.
Washington opposes any move by UN bodies to recognise the Palestinian territories as a state, believing this must await a negotiated Middle East peace deal.
But President Donald Trump's administration is also reviewing many of its multilateral commitments, pursuing what he calls an "America First" policy that has allies in Europe worried.
"UNESCO is about promoting our ideals and values through culture, education and science," France's UN ambassador Francois Delattre told reporters in New York, adding that "we need an America that stays committed to world affairs."
But Nauert said that the cost to the United States of remaining a member had also influenced the decision.
"As many of you know, we were in arrears to the tune of $550 million or so. And so the question is, you know, do we want to pay that money? And do we want to pay more money going forward," she said.
