The ruling involving his 2017 convictions and sentence closed a grim chapter in European history that included the 1995 Srebrenica massacre of more than 8,000 Muslim men and boys.
Presiding Judge Prisca Matimba Nyambe of Zambia said the panel had dismissed, by a vote of 4-1, his appeals of convictions for crimes including genocide, murder, extermination and terror for atrocities throughout the war.
The 79-year-old former general is the last major figure to face justice from the conflict that ended more than a quarter century ago.
His former political chief, ex-Bosnian Serb President Radovan Karadzic, already is serving a life sentence after being convicted for the same crimes.
Former Yugoslav President Slobodan Milosevic, who was accused of fomenting the ethnic conflicts that tore apart the Balkans in the 1990s, died in a UN cell in 2006 before judges at his trial could reach verdicts.
Serge Brammertz, the prosecutor who finally brought both Karadzic and Mladic to justice, said Mladic “ranks among the most notorious war criminals in modern history”.
“Mladic should be condemned by all responsible officials in the former Yugoslavia and around the world,” Mr Brammertz said.
“His name should be consigned to the list of history's most depraved and barbarous figures.”
US president Joe Biden said the “historic judgment shows that those who commit horrific crimes will be held accountable”.
The court also rejected an appeal by prosecutors of Mladic's acquittal on one other count of genocide linked to ethnic purges early in the war.
As commander of the Bosnian Serb Army, Mladic led troops responsible for atrocities ranging from ‘ethnic cleansing’ campaigns to the siege of Sarajevo and the war's bloody climax in the 1995 Srebrenica massacre.
In Sarajevo, applause broke out among those watching the proceedings. Mayor Benjamina Karic called it “a day of justice” for Sarajevo, Bosnia and innocent victims of the war.
Nedziba Salihovic, who lost her son and husband in the bloodshed, watched the court hearing on a large screen in Srebrenica.
“This means a lot to me, my heart is racing,” she said. “He was punished. It is not important where he'll end up [to serve his sentence].
“Like mothers of Srebrenica, he'll spend the rest of his life without his family.”
Additional reporting by the Associated Press.