“Woman, life, freedom” and “death to the dictator” chanted dozens of men and women at the Aichi cemetery in Saqez, Amini's hometown in the western Kurdistan province, in videos shared online.
The 22-year-old Iranian of Kurdish origin died three days after being taken into custody by the notorious “morality police” on September 13 while visiting Tehran with her younger brother.
Since then, protests have flared across the country in the largest display of public anger at the government in at least three years.
Young women and schoolgirls have led the charge, removing and burning their headscarves and confronting security troops on the street.
Wednesday marks 40 days since Amini's death and the end of the traditional mourning period in Iran.
Mourners headed to her grave to mark the occasion even though the security services had warned her family not to hold the ceremony, saying that otherwise “they should worry for their son's life”, according to activists.
It is unclear if Amini’s family took part. State news agency Irna published a statement it said was from them, saying that “considering the circumstances and in order to avoid any unfortunate problem, we will not hold a ceremony marking the 40th day”.
Activists said the statement was made under pressure.
Images shared by the Hengaw rights group showed the heavy presence overnight of security forces in Saqez. They reportedly shut off entrances to the city.
Despite that, dozens of people were seen streaming into the city in cars and on motorcycles, as well as on foot along roads and across open fields, in footage posted online by Hengaw.
“Kurdistan, Kurdistan, the graveyard of fascists,” a group of them were heard chanting, according to AFP news agency in another video shared by activists on Twitter.
“The cities of Sanandaj, Saqez, Divandarreh, Marivan and Kamyaran are on widespread strike” in Kurdistan province, Hengaw said on Twitter.
Security forces opened fire at mourners in Amini's Kurdish home town of Saqez on Wednesday, a witness said.
"Riot police shot mourners who gathered at the cemetery for Mahsa's memorial ceremony … dozens have been arrested," the witness said. Iranian authorities were not available to comment.
There were separate reports that oil refinery workers also walked out on Wednesday in a further blow to one of the country’s most crucial industries.
The rights group, which monitors rights violations in Kurdistan, said Iranian football stars Ali Daei and Hamed Lak had travelled to Saqez “to take part in the 40th-day funeral” and were staying at the Kurd Hotel.
But they “had been taken to the government guesthouse … under guard by the security forces”, Hengaw said.
Daei has previously run into trouble with authorities over his online support for the Amini protests.
Unverified footage posted by the Oslo-based group Iran Human Rights showed people gathering outside the Kurd Hotel in Saqqez “in their night protests”.
Hammihan newspaper later quoted Kurdistan governor Esmail Zarei Koosha as saying that Daei and other celebrities were in Tehran and that “everything is calm in Saqez”.