The ex-mayor, Daniel Ceballos, had been held at the Helicoide, the Sebin intelligence service's detention site in the capital Caracas. Critics say the site is used by Maduro to crack down on his opponents.
"You are the first group to benefit from these measures," said assembly leader Delcy Rodriguez, a former foreign minister, adding that other releases would follow on Saturday and in the following days.
The Supreme Court of Justice said a total of 39 prisoners were being released Friday, though not all were opponents of Maduro.
Some of them had been involved in beating up an opposition deputy during the campaign for recent presidential elections won by Maduro.
Ceballos was one of several inmates who in May briefly took over a part of the Helicoide in protest ahead of the election that saw Maduro win a second term.
As a result, dozens of prisoners were transferred to other sites and four minors were freed.
It remains unclear how the inmates took control of the space as they were not armed, nor how they could send videos and photos of what happened.
"It was President Maduro who said, after his election victory on May 20, 'this is the way, the path of dialogue, of union and of peace,'" Rodriguez said.
Rodriguez was speaking at a ceremony marking the release, where she was flanked by Attorney General Tarek William Saab and three opposition governors who she said had "initiated a process of dialogue" with Maduro, but gave no details.
"We can think differently I know, but we can understand each other through means other than violence," she said.
'Pacification process'
Communications Minister Jorge Rodriguez told a press conference that Maduro hopes his decision to release the prisoners "will lead to a process of pacification."
Ceballos had posted on Twitter a picture of a group of prisoners that included American Joshua Holt, who has been accused of espionage but was recently released and sent home to the United States.
San Cristobal, a city in western Venezuela near the border with Colombia, was a flashpoint in a wave of deadly anti-government protests in 2014.
Ceballos was first arrested in March 2014, then moved to house arrest and sent back to prison in 2016. He stood accused of inciting violence.
The chairman of the country's highest court, Maikel Moreno, said Ceballos would have to report to the authorities every 30 days, and was subject to a ban on leaving the country and making statements to the media and on social networks.
Under Maduro, Venezuela is going through the worst economic crisis in its history.
Hyperinflation has crippled the country, leading to shortages of food and medicine. Hundreds of thousands of people have fled Venezuela to escape the growing deprivation.
Upon his re-election, Maduro promised to work for reconciliation and offered to free political opponents who have not committed serious crimes, in order to "overcome the wounds" of the protests against him, which have left some 200 dead since 2014.
According to rights group Foro Penal, there are about 350 political prisoners in Venezuela -- a figure the Maduro government disputes.
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