"Those who draw swords against the regime will be confronted," Khamenei, who has the final say on all national issues, said as he led the weekly Muslim prayers at Tehran university.
"Differences of views should not lead to conflicts … the policy of the regime is to work with the majority. But if opposition groups have ideas that are against the nation's security and the principles of the regime, they will be confronted," he said.
The Islamic republic is engulfed in its worst crisis following the disputed re-election of President Mahmoud Ahmadinejad in the June 12 poll, which his rivals claim was the result of massive vote rigging.
The all-powerful Khamenei has steadfastly defended Ahmadinejad's victory.
Khamenei also said Iran was not afraid of Western powers, especially the United States and Britain.
"The US government and the British government have two hundred years of wicked attitude towards Iran. So be it. This will not intimidate anyone," the country's top spiritual guide said as worshippers chanted: "Death to America! Death to Israel!."
The university was packed with worshippers and black-clad supporters of Khamenei who shouted "We are here because of our love for the leader" and "The hand of God is on our head…Khamenei is our leader."
Some worshippers carried Khamenei posters on which was written: "Oh our free leader. We are ready, we are ready."
Top Iranian officials and clerics were present to hear the sermon, including Ahmadinejad, parliament speaker Ali Larijani and chief of judiciary Sadeq Larijani.
Since his June address at a time when Tehran was rocked by anti-Ahmadinejad protests, Khamenei has regularly defended the president.
On June 19, Khamenei ruled out any major fraud in the presidential poll and warned that defeated candidates would be held accountable over the street violence.
Last Friday, deputy parliament speaker Mohammad Reza Bahonar said Khamenei had urged MPs to approve most of Ahmadinejad's proposed cabinet members during a vote of confidence the previous day.
