The European Union on Sunday told Iran to reconsider its decision to withdraw accreditation from several inspectors from the United Nations nuclear watchdog.
The International Atomic Energy Agency had warned Saturday that the "disproportionate and unprecedented" move would seriously hamper its work.
Brussels, which acts as coordinator for the 2015 nuclear deal between Tehran and world powers known as the JCPOA, said it was "highly concerned" by the development.
"Particularly worrying is the direct and severe impact of this decision on the agency's ability to conduct its verification activities, which includes the monitoring of the Joint Comprehensive Plan of Action [JCPOA]," a spokesman said.
"The EU urges Iran to reconsider its decision without delay."
Iran's foreign ministry said Saturday that the move was in retaliation for "political abuses" by the United States, France, Germany and Britain.
In 2015, major world powers reached the accord with Iran under which Tehran would curb its nuclear programme in exchange for relief from crippling economic sanctions.
Efforts to revive the deal have been fruitless so far.