"You (Americans) want to negotiate when you are pointing the gun at Iran. The Iranian nation will not be intimidated by such actions," Khamenei told air force commanders, according to excerpts of his speech posted on his website.
"Some rejoice at the offer of negotiations ... (but) negotiations will not solve anything," he said.
The all-powerful Khamenei has final say on all key issues in the Islamic republic, including Iran's sensitive nuclear activities and foreign policy.
"I am not a diplomat but a revolutionary," he said.
"The US policies in the Middle East have failed and the Americans are in need of a winning hand. That is bringing Iran to the negotiating table."
He made the remarks in response to an offer put forward by US Vice President Joe Biden to engage Iran directly to resolve the nuclear issue provided "the Iranian leadership, supreme leader, is serious."