State Department spokeswoman Heather Nauert said Washington would establish an "observer mission" to replace its representation at the Paris-based agency.
The head of UNESCO Irina Bokova voiced "profound regret" over the decision by the United States and called it a "loss to multilateralism".
"I wish to express profound regret at the decision of the United States of America to withdraw from UNESCO," Bokova said in a statement.
The United States was angered in 2011 when UNESCO members granted Palestine full membership of the body, despite opposition from its ally Israel.
Washington opposes any move by UN bodies to recognize the Palestinians as a state, believing that this must await a negotiated Middle East peace deal.
But US President Donald Trump's administration is also reviewing many of its multilateral commitments, pursuing what he calls an "America First" foreign policy.
Nauert said the State Department had notified UNESCO's outgoing Director-General Irina Bokova of their decision earlier Thursday.
"This decision was not taken lightly, and reflects US concerns with mounting arrears at UNESCO, the need for fundamental reform in the organization, and continuing anti-Israel bias at UNESCO," she said in a statement.
"The United States indicated to the director-general its desire to remain engaged with UNESCO as a non-member observer state in order to contribute US views, perspectives and expertise on some of the important issues undertaken by the organization, including the protection of world heritage, advocating for press freedoms and promoting scientific collaboration and education."