Samantha Power accused African countries and their supporters of latching on to a procedural argument to cover up their anti-gay bias.
It was the second time that the African group had tried to push through a measure demanding talks on the mandate of the expert who will report on abuses against lesbian, gay, bisexual and transgender (LGBT) people worldwide.
The measure was defeated by a vote of 84 against to 77 in favor, with 16 abstentions.
European countries and the United States had lobbied energetically in the 193-nation assembly to defeat the African push to suspend the appointment of the expert.
The UN Human Rights Council in September appointed international law professor Vitit Muntarbhorn of Thailand to investigate cases of discrimination and violence against LGBT people worldwide.
Burkina Faso argued on behalf of the African countries that there was no legal basis for the mandate and no international recognition of sexual orientation and gender identity as human rights.
US Ambassador Samantha Power accused African countries and their supporters of latching on to a procedural argument to cover up their anti-gay bias.
"This is not an issue of the North trying to impose its values on the South. It is an issue of respecting the dignity and human rights of all people everywhere," Power told the General Assembly.
Dutch Ambassador Karen van Osteroom, whose country has adopted progressive policies on LGBT rights, said the United Nations must shine a light on anti-gay violence.
"People around the world are being bullied, are being jailed, are being beaten, are being killed, for no other reason than for which gender they identify with most, or for whom they happen to love," he said.
"And that is what the mandate on the independent expert is all about," he said.
African countries failed last month to push for a similar measure during a vote in the assembly's human rights committee.
A total of 73 countries -- almost 40 percent of all 193 UN members -- still have laws on their books making homosexuality a crime.
In Africa alone, 33 countries have anti-gay laws including Uganda, Nigeria, Sudan and Mauritania.
Muntarbhorn, who began work as the UN expert last month, has been appointed for three years.
He will carry out country visits, raise allegations of LGBT rights violations with governments and work to protect rights defenders.