The EU’s high representative for foreign affairs, Josep Borrell, on Friday urged Iran to stop the repression of protesters and to release those who have been detained.
In a tweet, Mr Borrell said he had conveyed his message to Iranian Foreign Minister Hossein Amir Abdollahian.
“I spoke with @Amirabdolahian to convey again EU's clear and united position: people in Iran have the right to peaceful protest and to defend fundamental rights,” Mr Borrell said.
“Violent repression must stop immediately. Protesters must be released. Internet access and accountability are needed.”
Iranian activists on Friday called for fresh nationwide protests over the death of Mahsa Amini, 22, on September 16.
She had been in the custody of Iran’s morality police, who held her for wearing her headscarf “improperly”.
Outrage over her death has fuelled the biggest wave of street protests and violence seen in the country for years, though observers do not believe that the unrest is close to toppling the government.
At least 108 people have been killed in the protests, and at least 93 more have died in separate clashes in Zahedan, capital of the south-eastern province of Sistan and Baluchestan, according to Oslo-based group Iran Human Rights.
There were fewer reports of people taking to the streets over Amini's death on Friday, but hundreds of men were seen protesting after weekly prayers in Zahedan.
Despite blocked access to internet services and platforms like Instagram and WhatsApp, activists issued an online appeal for a huge turnout on Saturday for protests under the slogan, “The beginning of the end!”
They have called on people across Iran to show up at spots where the security forces are not present and to chant, “Death to the dictator”.
“We have to be present in the squares, because the best VPN these days is the street,” activists said, referring to virtual private networks used to circumvent internet restrictions.
The bloody crackdown has drawn international condemnation and new sanctions on Iran from Britain, Canada and the US.
Iran's supreme leader Ayatollah Ali Khamenei has accused the country's enemies of fomenting riots.
On Friday, his government criticised French President Emmanuel Macron for expressing solidarity with the protests.
Foreign ministry spokesman Nasser Kanani said Mr Macron's remarks were “meddlesome” and served to encourage “violent people and law breakers”.
Mr Kanani said it was “surprising” that France was condemning Iran's security forces for dealing with “violent people and rioters”, when it was threatening to use force in response to “labour strikes in the oil and gas sector” at home.
“This is clear hypocrisy,” he said.
The French government moved this week to end strike in its refineries that has led to crippling shortages.
France's foreign minister said on Tuesday that five of its nationals were being held in Iran.
Agencies contributed to this report