In a frequently confrontational debate, Mr Milosevic constantly challenged the witness, Mahmut Bakalli - former Communist leader of the Yugoslav province of Kosovo - over the truth of his testimony.
"
It appeared to be an imposed apartheid which is a crime against humanity
"
Prosecution witness Mahmut Bakalli
Among others, Mr Milosevic queried Mr Bakalli's allegation that the former Yugoslav leader had known about the 1998 killing of 40 members of a single family - the Jasharis - in the Kosovo village of Prekaz.
Mr Milosevic said Jashari family members had refused to surrender, but Mr Bakalli retorted that women and children had been slaughtered.
Mr Bakalli is the first prosecution witness to testify against Mr Milosevic, who ended his cross-examination after about three-and-a-half hours.
Lively exchanges
The BBC's Peter Biles says Mr Milosevic started in a confrontational mood, but was confident while the witness appeared at times ill at ease.
Mr Bakalli had earlier told the court of what he called Mr Milosevic's policy of "apartheid" against the Albanian majority in Kosovo.
Milosevic charges
"You don't need to remind me of that," Mr Bakalli said.
Demanding a "yes or no" answer, Mr Milosevic then launched into detailed questioning of Mr Bakalli's role when he was Kosovo leader as well of his alleged links to the Kosovo Liberation Army - the armed group that began fighting for an independent Kosovo in the late 1990s.
Click here for extracts from Milosevic's defence
Mr Bakalli rejected this saying he had only had contacts of a political or diplomatic nature.
Both men were often interrupted by presiding Judge Richard May, who asked Mr Milosevic to give the witness a chance to respond and to stick to the line of questioning and Mr Bakalli to keep his answers short.
Mr Milosevic in turn asked Mr Bakalli about alleged anti-Serb bias in Kosovo's schools, a question which also received an abrupt response from the witness.
"You imposed the curriculum," Mr Bakalli said.
"You imposed the heads of the schools and the faculties."
First witness
Mr Bakalli was the first of up to 350 witnesses called by the UN to give evidence. He took the stand after Mr Milosevic concluded his three-day opening address.
In his testimony, Mr Bakalli, who said he had met Mr Milosevic while trying to calm tension after clashes between Serb security forces and ethnic Albanian guerrillas, testified he had suffered simply by being an Albanian.
Mr Bakalli, now a member of the new Kosovo parliament, said a Serbian security officer told him in 1997 that Mr Milosevic already had a "scorched earth" plan for Kosovo, which allegedly included the levelling of 700 ethnic Albanian settlements.
Mr Milosevic faces charges of genocide in Bosnia, and of crimes against humanity in Kosovo and Croatia.
He rejects the legality of the court and has refused to appoint lawyers to defend him in what is being described as the most important war crimes trial since the Nuremberg trials after World War II.
He is the first former head of state to be indicted before an international tribunal.