Khamenei also said the balloting had not been rigged, and he sided with hard-line President Mahmoud Ahmadinejad, offering no concessions to the opposition. He effectively ruled out any chance for a new vote, lauding the June 12 election as an expression of the people's will.
"Some of our enemies in different parts of the world intended to depict this absolute victory, this definitive victory, as a doubtful victory," Khamenei said at a Friday prayer service at Tehran University attended by tens of thousands of people. "It is your victory. They cannot manipulate it."
The speech created a stark choice for candidate Mir Hossein Mousavi and his supporters: Drop their demands for a new vote or take to the streets again in blatant defiance of the man endowed with virtually limitless powers under Iran's constitution.
Pro-Mousavi Web sites had no immediate reaction to Khamenei's warning. They did not announce changes in plans for a march at 4 p.m. Saturday from Revolution Square to Freedom Square, site of a massive rally Monday that ended with fatal clashes between protesters and a pro-government militia.
"We are all feel a little angry, worried and disappointed after the speech," said one Mousavi supporter, responding by e-mail to The Associated Press.
"We are waiting for Mousavi's reaction. He is our hope to protect our votes," added the Tehran resident, who spoke on condition of anonymity for fear of government retaliation.
Monday's demonstration was followed by three consecutive days of protest that have posed the greatest challenge to Iran's Islamic ruling system since the 1979 Islamic Revolution that brought it to power.
So far, the government has not stopped the protests with force despite an official ban on them. But Khamenei opened the door for harsher measures.
"It must be determined at the ballot box what the people want and what they don't want, not in the streets," he said. "I call on all to put an end to this method."
And Khamenei added, according to Press TV, Iranian state television's English-language channel: "Extremism in the country, any extremist move, will fan another extremist move. If the political elite want to ignore the law or break the law then they are taking wrong measures, which are harmful, and they will be held accountable for all the violence, bloodshed and rioting."
He accused foreign media and Western countries of trying to create a political rift and stir up chaos in Iran. Iranian leaders often blame foreign "enemies" for plots against the country, but Khamenei's comments suggest Iran could remain cool to expanding dialogue with the West and the offer of opening talks with Washington.
The House of Representatives voted overwhelmingly Friday to condemn Tehran's crackdown on demonstrators and the government's interference with Internet and cell phone communications.
The resolution was the strongest message yet to Iran from the U.S. government and was initiated by Republicans as a veiled criticism of President Barack Obama, who has taken a cautious line on the election dispute, expressing sympathy with protesters but avoiding condemnation of the Islamic government.
He said Tuesday that opposition to Ahmadinejad represented "a questioning of the kinds of antagonistic postures towards the international community that have taken place in the past, and that there are people who want to see greater openness and greater debate and want to see greater democracy."
Khamenei reacted strongly, saying Obama's statements contradicted the president's stated goal of opening dialogue with Iran and the conciliatory tone of other recent American messages.
"The U.S. president said 'We were waiting for a day like this to see people on the street,"' Khamenei said. "They write to us and say they respect the Islamic Republic and then they make comments like this. ... Which one should we believe?
Khamenei remained staunch in his defense of Ahmadinejad, saying his views were closer to the president's than to those of former President Hashemi Rafsanjani, a powerful patron of Mousavi.
Ahmadinejad watched the sermon from the front row and conservative candidate Mohsen Rezaei could be seen in the audience. State television did not show Mousavi in the crowd of thousands, which spilled out of the open-sided campus pavilion and filled surrounding streets.
Iran's Arabic-language state TV channel said before the service that Mousavi, Rezaei and reformist candidate Mahdi Karroubi would attend. Karroubi confirmed that but it was not clear from broadcasts of the sermon if he or Rafsanjani were in fact there.
Khamenei said the 11 million votes that separated Ahmadinejad from his top opponent, Mousavi, were proof that fraud did not occur.
"If the difference was 100,000 or 500,000 or 1 million, well, one may say fraud could have happened. But how can one rig 11 million votes?" Khamenei asked.
Khamenei said Iran would not see a second revolution like those that transformed the countries of the former Soviet Union and pointed a finger at the U.S., Britain and what he called Iran's other enemies.
British Prime Minister Gordon Brown and other European Union leaders expressed dismay over the threat of a crackdown. The British Foreign Office told Iran's charge d'affairs in London that Khamenei's comments were "unacceptable and had no basis in fact," a spokesman said on condition of anonymity in line with policy.
The Foreign Office summoned the Iranian ambassador but said that in the end, the more junior diplomat attended the meeting with political director Mark Lyall Grant.
In Switzerland, Iranian Nobel Peace laureate Shirin Ebadi said Iran should hold a new election observed by international monitors, adding that more than 500 people have been arrested since the balloting. Her human rights office in Iran was raided last year, its files confiscated and several members subsequently arrested.
Khamenei's address was his first since hundreds of thousands of Mousavi supporters flooded the streets, evoking the revolution that ended Iran's U.S.-backed monarchy. On Thursday, supporters dressed in black and green marched in downtown Tehran in a somber, candlelit show of mourning for those killed in clashes since the election.
Khamenei said the street protests would not have any impact.
"Some may imagine that street action will create political leverage against the system and force the authorities to give in to threats. No, this is wrong," he said.
The supreme leader left open a small window for a legal challenge to the vote. He reiterated that he has ordered the Guardian Council, an unelected body of 12 clerics and Islamic law experts close to the supreme leader, to investigate voter fraud claims.
The council has said it was prepared to conduct a limited recount of ballots at sites where candidates claim irregularities.
Ahmadinejad has appeared to take the growing opposition more seriously in recent days, backtracking Thursday on his dismissal of the protesters as "dust" and sore losers.
The crowds in Tehran and elsewhere have been able to organize despite a government clampdown on the Internet and cell phones. The government has blocked certain Web sites, such as BBC Farsi, Facebook, Twitter and several pro-Mousavi sites that are vital conduits for Iranians to tell the world about protests and violence.
Text messaging, a primary source of spreading information in Tehran, has not been working since last week, and cell phone service in Tehran is frequently down. The government also has barred foreign news organizations from reporting on Tehran's streets.
The BBC said it was employing two new satellites to help circumvent Iranian jamming of its Persian-language service.
Google said it was launching a Persian-to-English translation service and Facebook said Iranian users could now use a Persian version of its site as a way of easing communication to the outside world.
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