The industry, mining and trade ministry had previously allowed 24 models manufactured by the General Motors-owned Chevrolet to be brought in via a third country.
"Import order placement for American Chevrolet cars has been disabled since Sunday" on the government's online imports website, Farhad Ehteshamzadeh, head of the Association of Auto Importers, told the Fars news agency.
"After Chevrolet order placements were removed from the system, no orders will be allowed for (Chevrolet) cars," he said, without giving figures for the number of vehicles imported previously.
"Americans themselves don't use US-made cars," Khamenei said on Wednesday during a speech related to Labour Day on the importance of domestic production.
"We have seen this reflected in American media. They argue that fuel consumption is high and the cars are heavy."
If this is the case, why import vehicles from America, Khamenei asked.
"Who should stand against them? Officials themselves, respected ministers."
The United States and Iran have no diplomatic relations since 1980.
The deal came into force in January, lifting international sanctions in return for limitations on Tehran's nuclear programme to ensure it cannot manufacture an atomic bomb.
There are very few American cars in Iran other than those made before the 1979 Islamic revolution.
US-made vehicles can be bought in free trade zones, but their owners face restrictions on where they can be driven.
Since the nuclear accord, Tehran has been in talks to set up joint manufacturing plants inside Iran with major European and Asian companies including Fiat, Mercedes Benz, Peugeot, Renault and Volkswagen.
It increased to 1.1 million in 2014, and Iran is now hoping to make 1.6 million cars in 2018 and two million in 2022.
In November, before the sanctions were lifted, Nematzadeh also banned the import of US consumer goods to "prohibit products that symbolise the presence of the United States in the country" and to "boost national production".
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