The UN rights office said it was "deeply concerned" over the "zero tolerance" policy introduced by the administration of US President Donald Trump in a bid to deter illegal immigration.
Spokeswoman Ravina Shamdasani said information received from US civil society groups indicated that several hundred children had been separated from their parents at the border since October, including a one-year-old.
"The US should immediately halt this practice," she told reporters in Geneva.
In Washington, the White House made no immediate comment on the UN statement. But in a tweet, Trump, who demanded the separation policy be enforced at the beginning of May, blamed opposition Democrats for it.
"Separating families at the Border is the fault of bad legislation passed by the Democrats. Border Security laws should be changed but the Dems can't get their act together!," he said.
And Attorney General Jeff Sessions, the Trump administration's top law enforcement official, said the policy was legal and necessary.
"If people do not want to be separated from their children, then they should not bring them with them," he added.
Trump is under attack at home and internationally over the policy, which aims to stifle illegal immigration, especially by people fleeing violence in Central America who arrive at the border and immediately turn themselves in to officials, requesting asylum.
In the past, the applications were mostly accepted for review and the applicants, individuals and families, were released into the United States to wait for a decision.
- Policy a 'serious violation' -
In Early May, however, Sessions announced that all those detained for crossing the border without papers, including asylum seekers, would be immediately placed under arrest and charged with a crime.
Shamdasani condemned the separations as a human rights violation.
"The practice of separating families amounts to arbitrary and unlawful interference in family life, and is a serious violation of the rights of the child," she said.
"The use of immigration detention and family separation as a deterrent runs counter to human rights standards and principles," she said.
"The child's best interest should always come first, including over migration management objectives or other administrative concerns," she said.
The US says the policy aims to stem a surge of poor families mostly from Guatemala, El Salvador and Honduras.
Citing the daily violence in their home countries, thousands each week cross the US-Mexico border and ask for asylum.
In an interview with radio host Hugh Hewitt on Tuesday, Sessions defended the policy.
"You can't be giving immunity to people who bring children with them, recklessly, and improperly, and illegally," he said.
"The law requires us to keep children in a different facility than one for adults."