Iran's main cultural centre in the Sudanese capital was padlocked on Sunday, an AFP correspondent reported, and officials said the decision was final.
The Iranian flag still flew above the building on one of Khartoum's main roads across from the airport.
But an AFP reporter found the gate padlocked and no sign of anybody inside.
Tehran last week denied the centre and others had been shut on orders by Khartoum, despite generally close relations between the two countries.
A Sudanese official, speaking on condition of anonymity, said Thursday that the centers would close their doors by Sunday.
Sudan's foreign ministry told the centers to stop work and ordered their Iranian staff to leave the country.
"The decision to close the Iranian cultural centre is final and there will be no retreat from that," foreign ministry spokesman Yousif Al-Kourdafani said, quoted Sunday by the official SUNA news agency.
Sudan said it acted in response to the centers’ spreading of Shiite Islam, Iran's majority faith.
Sudan is overwhelmingly Sunni.
A Sudanese analyst told AFP the move by Khartoum might be in response to pressure from Riyadh, which further isolated the indebted and sanctions-hit Sudanese economy earlier this year by denying access to major Saudi Arabian banks.
Iranian warships have periodically made stops in Port Sudan, across the Red Sea from Iran's regional rival Saudi Arabia.
The 25-year-old regime of President Omar al-Bashir relies on a base of support that is essentially the same as the Muslim Brotherhood, which Saudi Arabia has declared a "terrorist" organization.
The group is also banned in many Gulf Arab countries.
Newspaper columnist Abdalla Rizig Abu Simazeh said Sudan will expect the Gulf Arab countries to reward its action against the Iranians.
But closing the cultural centers will not have much effect on broader Sudanese-Iranian ties and so "will probably minimize its impact in restoring Sudan's relations with the Arab Gulf countries and Egypt", Simazeh wrote in Sunday's The Citizen.
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