Washington expressed "regret" at losing the right to decide matters in the 195-member Paris-based UN agency, which resulted when the US congress activated an automatic suspension of payments over the Palestinian accession.
"We regret that today the United States lost its vote in the United Nations Educational, Scientific and Cultural Organisation (UNESCO) General Conference," State Department spokeswoman Jen Psaki said in a statement.
She noted however that the abandonment of its voting right "is not a loss of US membership," and said the United States intends to continue its active participation.
Psaki said US President Barack Obama had asked Congress for "legislative authority to allow the United States to continue to pay its dues to UN agencies that admit the Palestinians as a member state when doing so is in the US national interest."
Under UNESCO rules, the US and Israel had until Friday to pay their dues or automatically lose voting rights.
US contributions represented 22 percent of the agency's overall budget. With the US and Israel withdrawing their contributions, UNESCO's budget fell from $653 million to $507 million (310 million euros).
The reduced budget means some 300 people at UNESCO are in danger of losing their jobs. In 2012 the UN agency employed 1,200 people at its headquarters in Paris and 900 around the world.
"The list of countries that will lose their voting rights will be announced probably tomorrow (Saturday) in a plenary session," the same UNESCO source said.
UNESCO's chief Irina Bokova has raised $75 million to deal with the financial crisis at the agency, which is responsible for selecting and overseeing World Heritage sites and deals with literacy, media freedom, science and environmental issues.
In October the 61-year-old Bulgarian was re-elected as UNESCO's director general after a tough challenge from two rivals -- the Djiboutian ambassador to France, Rachad Farah, and Lebanese academic Joseph Maila.
Both Farah and Maila argued the body had lost its main objective to maintain world peace through culture.
UNESCO chief concerned over smaller budget
In the run-up to the election, Bokova, a former foreign minister, said the financial crisis at the agency was over and argued the body had been able to maintain its programmes.
But she did express concern over the tighter budget, saying it was "a derisory amount in respect of our mandate".
France's Court of Auditors charged with conducting financial and legislative audits of most public institutions, has been scathing in its criticism of UNESCO saying the "unexpected nature (of the fund freeze) could not justify the organisation's state of unpreparedness".
Other reports however, notably by Britain and Australia, had voiced general approval of Bokova's tenure.
The Palestinians were admitted to UNESCO in October 2011, becoming the agency's 195th member when its general assembly voted 107-14 in favour of the membership bid.
The United States says the Palestinians must reach a peace agreement with Israel before they can become full members of an international organisation.