The "secret door" of the Sidi Yahia mosque in the fabled caravan city fell victim to a spree of destruction in 2012 by Al-Qaeda-linked Ansar Dine, one of several radical Islamist groups which seized key northern cities that year.
Around 100 Malian political and religious leaders, diplomats and representatives from world heritage body UNESCO gathered for a ceremony, held with a heavy security presence, an AFP journalist at the scene reported.
"This is a very important day," said the mosque's imam, Alphadi Wandara. "Since (the days) of our forefathers, for centuries the door has been like that: closed."
UNESCO craftsmen had spent five months restoring the doors this year.
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Former teacher Ahmad Al-Faqi Al-Mahdi, who took part in wrecking Timbuktu's holy shrines as well as the mosque's doors, is currently awaiting a sentence on war crimes charges in The Hague for his actions.
Sidi Yahia was among several sites in Timbuktu attacked by Ansar Dine, which considered the city's mausoleums idolatrous according to their strict interpretation of Islam.
Timbuktu youth leader Salah Maiga told AFP he had witnessed the jihadists' campaign with his own eyes.
"We were there with these barbarians, these lawless people without religion, who claim to speak in the name of Islam," Maiga added, saying he hoped for justice in Mahdi's case.
Mahdi has admitted the charges against him and prosecutors are asking for a jail term of 11 years at his sentencing on September 27.
Ansar Dine picked out Mahdi to head its "Hisbah" brigade, which policed infractions of Islamist behaviour in Timbuktu.
Revered as a centre of Islamic learning during its golden age in the 15th and 16th centuries, Timbuktu in northern Mali is also known as the "Pearl of the Desert".