The remarks came after a year in which Iran-backed Hezbollah in Lebanon and Hamas in Gaza suffered heavy losses in wars with Israel, and two weeks after the fall of Syrian president Bashar al-Assad, who had been a key link in Tehran's so-called axis of resistance.
Another spoke of that axis, Yemen's Huthi rebels, have been repeatedly targeted by the United States and Britain over their attacks on Red Sea shipping lanes, launched in solidarity with Palestinians.
"The Islamic Republic does not have a proxy force. Yemen fights because it has faith. Hezbollah fights because the power of faith draws it into the field. Hamas and (the Islamic) Jihad fight because their beliefs compel them to do so. They do not act as our proxy," supreme leader Ayatollah Ali Khamenei told a group of visitors in Tehran.
"They (the Americans) keep saying that the Islamic Republic has lost its proxy forces in the region! This is another mistake," he said, adding: "If one day we want to take action, we do not need a proxy force."
Earlier this month, Syrian rebels' lightning push to Damascus from their strongholds in the northwest ended the decades-long rule of Assad's family, which had been an ally of Tehran.
Khamenei predicted "the emergence of a strong, honourable group" in Syria, saying the country's young men had "nothing to lose."
"His university, school, home, street and life are insecure; what should he do? He must stand with strength and determination against those who have designed this insecurity and those who have implemented it, and God willing, he will overcome them."
Assad had long played a strategic role in Iran's anti-Israel axis of resistance, particularly in facilitating the supply of weapons to Hezbollah in neighbouring Lebanon.
The axis of resistance also includes Hamas, the Huthis and smaller Shia militia groups in Iraq.
All of the groups are united in their opposition to Israel and its main backer the United States.
The supreme leader, who has the final say in major state policies, also accused the United States of trying to create chaos and unrest in Iran.
"The Iranian nation will trample under its strong feet anyone who accepts America's mercenary role in this regard," he said.
