UNESCO is the first UN agency the Palestinians have joined as a full member since President Mahmoud Abbas applied for full membership of the United Nations on 23 September.
The United States, Canada, Germany and Holland voted against Palestinian membership. Brazil, Russia, China, India, South Africa and France voted in favor. Britain and Italy abstained.
Washington is likely to cut funding to UNESCO over the vote.
"The action today will complicate our ability to support UNESCO," David T. Killion, US ambassador to UNESCO, told journalists after the vote.
"The US has been clear for the need of a two-state resolution, but the only path is through direct negotiations and there are no shortcuts, and initiatives like today are counterproductive."
The vote highlighted divisions over foreign policy within the European Union, some of whose 27 members voted for and some against Palestinian membership.
Austrian UNESCO ambassador Ursula Plassnik, whose country voted in favor, said she regretted the European Union could not arrive at a common position on the Palestinian issue.
The Palestinians obtained backing from two thirds of UNESCO's members to become the 195th member of UNESCO, with status as "an observer entity." Of 173 countries that voted from a possible 185, 107 voted in favor, 14 voted against, 52 abstained and 12 were absent.
Forty representatives of the 58-member board has voted in favor of putting the matter to a vote earlier this month, with four -- the United States, Germany, Romania and Latvia -- voting against and 14 abstaining.
Admission will be seen by the Palestinians as a moral victory in their bid for full U.N. membership but could be costly for UNESCO.
US legislation stipulates that it can cut off funding to any UN agency that grants full membership to Palestinians.
Israel called the vote a "tragedy."
"This resolution is a tragedy for UNESCO. UNESCO deals in science and not science fiction and nevertheless (it) adopted the science fiction reality," said Nimrod Barkan, Israel's ambassador to UN organisation.
Israel has said the Palestinian bid would amount to politicization of the agency that would undermine its ability to carry out its mandate.