Americans will be expelled from Iraq and Syria, Iran's supreme leader Ayatollah Ali Khamenei said on Sunday.
Iran came close to conflict with the US when an American drone strike killed top Iranian military commander Qassem Suleimani in Baghdad on January 3.
Tehran retaliated with a missile barrage against an American base in Iraq days later.
"The Americans won't stay in Iraq and Syria and will be expelled," Mr Khamenei said.
Tehran has also summoned the Swiss ambassador, who represents US interests in Iran, over measures Washington could take after an Iranian ship delivered fuel to Venezuela, the Mehr news agency reported.
A senior official in US President Donald Trump’s administration said last week that it was considering its response.
The oil sectors of Iran and Venezuela, members of Opec, are under US sanctions.
The US official declined to specify the measures being considered but said options would be presented to Mr Trump.
Iran's deputy foreign minister, Abbas Araqchi, passed a message to the ambassador on Sunday, warning against any US threat to its tankers.
Iranian Foreign Minister Javad Zarif also wrote a letter to UN Secretary General Antonio Guterres warning that any American measures against the fuel shipment would be dangerous, illegal and a form of piracy, the foreign ministry website said.
At least one tanker carrying fuel loaded at an Iranian port has set sail for Venezuela, according to vessel tracking data from Refinitiv Eikon on Wednesday.
That could help to ease an acute scarcity of petrol in the South American country led by President Nicolas Maduro, who many nations consider to be illegitimate.