The onslaught of 59 cruise missiles followed a suspected chemical attack on the rebel-held town of Khan Sheikhun in northern Syria that killed 87 civilians, including many children.
Tehran has been a key backer of President Bashar al-Assad in Syria's six-year civil war, referring to all rebel groups in the country as "terrorists".
"The United States made a strategic mistake," Khamenei said.
"The Islamic Republic of Iran will not leave the field (...) in the face of threats," he said, alluding to suspicions that the United States intended the attack as a sign it was willing to attack other countries including Iran.
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