Pierre Claver Mbonimpa, a well known human rights activist, was shot in Bujumbura, Burundi by gunmen on motorbikes on Monday evening, his family members and witnesses said.
The activist was immediately taken to Bujumbura hospital after the attack.
"He was going home when suddenly a group of motorcyclists encircled him and started shooting at him. Many of the bikers had guns," a relative of Mr Mbonimpa told Reuters.
Mbonimpa is believed to be one of the very few people in Burundi's civil society opposed to the president who had not fled the country.
He had strongly criticised President Pierre Nkurunziza's bid for a third term in office.
Reporters say while Mbonimpa’s works for the rights of prisoners and others have brought him international acclaim, rumours of his death had been heard in the country before the attack took place.
Earlier, presidential aide Gen Adolphe Nshimirimana, who was in charge of the president’s personal security, was also shot in his car on Sunday.
Burundi has been hit by weeks of civil unrest as the country’s opposition wants Nkurunziza to withdraw his third-term presidential bid, which violates the country's constitution and the Arusha Accords, a peace deal that ended over a decade long ethnic civil war in 2005.
Burundi’s constitution limits the presidency to two terms in office, but Nkurunziza's supporters say he can run again because he was elected to his first term by lawmakers rather than the public.
Dozens of people have been killed and hundreds wounded since April, during protests opposing Nkurunziza’s decision to run for a third term.
More than 100,000 people have fled Burundi since the start of the crisis, the UN says.