The new rule will require same-sex couples to be married for their partners to receive a visa, a move that will increasingly distress those couples who are from countries where same-sex marriage is illegal.
The United States is denying visas to the partners of more than 100 gay diplomats unless they get married, officials said on Tuesday.
"It is an unfortunate change in rules, since same-sex couples, unlike opposite-sex couples, have limited choices when it comes to marriage", U.N. Globe, a group that advocates for LGBT staff at the U.N., said in a statement released before the change.
The change reverses a policy put in place in 2009 under former Secretary of State Hillary Clinton that defined family to include domestic partners of diplomats posted at embassies or the United Nations.
Exceptions may be made for those who come from countries that don't allow same-sex marriage but do accept USA same-sex diplomatic spouses for accreditation.
"State Dept. will no longer let same-sex domestic partners of United Nations employees get visas unless they are married", she tweeted, noting that "only 12% of United Nations member states allow same-sex marriage".
Married same-sex spouses - like opposite-sex spouses - will still be eligible to enter the country, but it remains illegal in most countries around the world for same-sex couples to Wednesday.
David Pressman, former USA ambassador to the U.N. Security Council in the Obama administration, slammed the rule reversal because the U.N.is "composed of probably one of the most diverse workforces of any organization in the world".
If they do not comply by the December 31 deadline, and subsequently submit proof of marriage, they are expected to leave the country within 30 days of the deadline.
"Only 12% of United Nations member states allow same-sex marriage", she wrote on Twitter. "In light of the landmark 2015 Supreme Court decision legalizing same-sex marriage, the USA extends diplomatic visas only to married spouses of US diplomats".
Heterosexual couples must now be married to obtain the G-4 visa.
Yet that exception was not offered to United Nations officials. But that government would have to provide documentation proving that same-sex marriage was illegal and commit to accepting same-sex partners of USA diplomats.
But the policy could pose a major problem for some diplomats and employees because in many countries, same-sex marriage still isn't allowed, with less than 15 percent of countries in the world recognizing it as legal.
There are now 71 countries that criminalise same-sex relations, according to the International Lesbian, Gay, Bisexual, Trans and Intersex Association (ILGA).