Khamenei's statement, reported on Iranian television, comes before a meeting between EU foreign policy chief Javier Solana and Iran's top nuclear negotiator, Saeed Jalili, scheduled for July 19, when they are due to discuss the format of further nuclear talks.
The supreme leader said Iran would not back down from the "red line" of continuing uranium enrichment for civilian purposes.
"Iran made the decision to participate in talks [on its nuclear program], but these talks will be successful only if no threats are voiced," he said. "The Iranian people do not accept any threats."
He also warned against aggression against his country. "If the U.S. or Israel chooses this silliness, let them know: the Iranian nation will cut off the aggressor's hand," he said.
Khamenei added that the Iranian people would punish President George Bush if he ordered the U.S. military to destroy Iran's nuclear infrastructure.
"Some say the current U.S. president, in the last months of his presidential term, will commit this action [an attack on Iran] to create a headache for the future American administration," Iranian TV quoted Khamenei as saying.
"But this is an erroneous opinion. If someone decided to take such actions, the Iranian people would pursue them and punish them even if they were no longer in power."
In recent months, international media have carried reports about a possible military attack by Israel and the United States on Iran's nuclear facilities.
Bush has repeatedly said he would not rule out any options for resolving the Iranian nuclear dispute, but that he would prefer diplomatic means.
Solana handed Iranian Foreign Minister Manouchehr Mottaki a package of new incentives from the Iran Six - China, France, Russia, the United States, Germany and Britain - on June 14. The proposals offer political, security and trade benefits to Iran in exchange for suspending its nuclear program.
However, the six world powers have demanded that Iran impose a moratorium on uranium enrichment prior to the talks on the issue.
Iran responded to the European Union early in July but said it had no plans to drop its nuclear enrichment.
Iran is currently under three sets of relatively mild UN Security Council sanctions for defying demands to halt uranium enrichment, which it says it needs purely for electricity generation despite Western accusations that the program is geared toward weapon production.
Iran maintains that it has never been involved in research into the development of nuclear weapons.
