The men were guilty of crimes against humanity, murder, persecution on political grounds and other inhumane acts over the forced evacuation of Phnom Penh in Cambodia after the fall of the city in 1975, the tribunal concluded.
Under the Marxist leader Pol Pot, the regime ruled Cambodia between 1975 and 1979, forcing millions of people from the cities to work on communal farms in the countryside to make the country self-sufficient.
Money, private property and religion were banned and anyone considered intellectual or middle class was tortured and executed.
The two former leaders, 85-year-old head of state Khieu Samphan and the right-hand man to Pol Pot, 90-year-old Nuon Chea were given life sentences in 2014 by a UN-assisted Khmer Rouge tribunal.
Upholding the sentences, the Supreme Court said the verdict was “appropriate”.
Tribunal spokesman Lars Olsen said: “It is a historic day for Cambodia. For the first time in 41 years someone in the national leadership has been held criminally responsible for the crimes committed by the Khmer Rouge regime.”
The two men, who were found guilty of crimes against humanity including extermination, enforced disappearances and political persecution, sat impassively as the lengthy verdict was read out.
The pair were detained in 2007 and started their sentences in 2014 inside the Khmer Rouge tribunal’s facility, where conditions are much better than ordinary Cambodian prisons and offer access to radio and television.
Khieu Samphan and Nuon Chea told the tribunal they were unfairly being singled out while the Cambodian government sought to block the tribunal from trying other suspects.
They also alleged a number of legal and factual errors as well as biases by the judges.
The men are still on trial in a second case where they are facing charges of genocide against ethnic minorities and foreigners, and implementing policies of rape and forced marriages.
All the charges were originally to have been part of one trial, but fears that they would die before proceedings could finish led to their case being broken into two parts, known as Case 002/01 and 002/02.
Their two co-defendants, Ieng Sary, the third-ranking Khmer Rouge leader and its foreign minister, and his wife, Ieng Thirith, died during the first phase of their trial.
Seak Ny, 64, whose husband died of starvation under the regime, said: “I waited for this moment for 40 years. It has now arrived.”
She said the Khmer Rouge also killed her older brother and his five children when they discovered he was a former soldier in the government they ousted.