The Americans should know that if they attack Iran, their interests will be harmed around the world, Khamenei said.
The Iranian nation will respond to any attack twice as strongly, he added.
In March, the United Nations Security Council adopted a Presidential Statement unanimously giving Iran until April 28 to suspend all of its uranium enrichment activities and resume its cooperation with the International Atomic Energy Agency (IAEA).
Irans hard-line President Mahmoud Ahmadinejad has since said that Tehran would never abandon its right to uranium enrichment.
Washington has called on the Security Council to impose sanctions on Tehran for failing to heed the demands of the UN nuclear watchdog.