Speaking to laborers on the occasion of the International Laborers Day, Khamenei said U.S. officials have been using threatening language against Iran for the past 27 years, but the Iranian nation and officials do not care about the threats.
"The Iranian nation and its officials are peace-seekers and the Islamic republic would not invade anybody," the television quoted Khamenei as saying.
But he added: "The Americans should know that if they invade Iran, their interests around the world would be harmed. Iran will respond double-fold to any attack."
Iran is locked in a dispute with the United States and its allies over its controversial nuclear program and is facing a U.N. deadline Friday to suspend uranium enrichment or risk possible sanctions. The tension and risk of future conflicts is affecting the international oil market.
Khameni, a former close confidant of Ayatollah Khomeini, is regarded as a key figurehead of Iran's conservative establishment.
Also Wednesday, Iranian President Mahmoud Ahmadinejad said that Iran's "enemies" would not be able to use the U.N. Security Council and the International Atomic Energy Agency to punish Iran.
"The enemies could not impose their wrong decision against us under cover of the Security Council and the IAEA," he said, according to the television broadcast.
Iran's top nuclear negotiator warned Tuesday the country would halt all cooperation with the U.N. nuclear watchdog if the Security Council imposes sanctions, and warned it might go further and hide its nuclear program if the West takes other "harsh measures."
The statements by Ali Larijani were Iran's strongest defiance yet of a Friday deadline, set by the Security Council, for Iran to suspend enrichment of uranium, a process that can produce fuel for nuclear reactors or material for warheads.
"Military action against Iran will not end our program," Larijani said Tuesday, speaking at a conference on the energy program. "If you take harsh measures, we will hide this program. If you use the language of force, you should not expect us to act transparently."
The comments surprised many of the international delegates at the conference, reports CBS News correspondent Elizabeth Palmer.
"That is a sign of a confidence here that may be misplaced — that they can both withstand sanctions and even a military strike," said conference delegate Rosemary Hollis of the Royal Institute of International Affairs.
U.S. Secretary of State Condoleezza Rice responded by saying Iran's statements were further isolating it from the international community. "Iranians can threaten, but they are deepening their own isolation," she said in Athens, where she was meeting with officials.
Ahmadinejad on Monday also renewed his criticism of Israel, calling it a "fake regime" that cannot continue to exist.
"Iran's threatening statements about Israel and its defiance are part of the reason that Security Council members are united about sending a message to Iran about its nuclear program," said CBS News foreign affairs analyst Pamela Falk at the United Nations. "But as the deadline at the U.N. approaches, action on sanctions appears unlikely."