He said on Friday that there was "definitive victory" and no rigging in the disputed presidential elections, offering no concession to protesters demanding the vote be canceled and held again.
He remained staunch in his defense of Ahmadinejad, saying his views were closer to the president's than to those of Hashemi Rafsanjani, a powerful patron of opposition leader Mir Hossein Mousavi.
In his first public address since demonstrators flooded the streets, Khamenei said protests should cease and the opposition must pursue its complaints within the confines of the cleric-led ruling system.
He said protesters would be held responsible for chaos if they didn't end days of massive demonstrations. The unrest has posed the greatest challenge to the system since the 1979 Islamic Revolution that brought it to power.
Khamenei said official results showing a landslide for hard-line President Mahmoud Ahmadinejad were beyond question.
"There is 11 million votes difference, Khamenei said. "How one can rig 11 million votes?"
"The enemies (of Iran) are targeting the Islamic establishment's legitimacy by questioning the election and its authenticity before and after (the vote)," FOX News quoted Khamenei, as saying.
Khamenei has already approved the June 12 election results that gave hard-line Ahmadinejad a landslide victory, but he has not been able to ignore the powerful defiance of the opposition, which has called the vote rigged, of his authority.
The address comes one day after hundreds of thousands of protesters in black and green flooded the streets of Tehran in a somber, candlelit show of mourning for those killed in clashes after Iran's disputed presidential election.
The supreme leader, who has the final say in all state matters, has tried to strike a compromise. On Monday, he ordered the Guardian Council, an unelected body of 12 clerics and Islamic law experts close to Khamenei, to investigate Mousavi's voter fraud claims. (ANI)