Ms Brnabic became prime minister in June this year and has tried to shift focus away from her sexual orientation, asking: "Why does it matter?"
Serbia's first openly gay prime minister joined several hundred activists at a gay pride march that was held amid tight security in the conservative Balkan country.
"That is my message today, that the Serbian government is here for all the citizens and that it will respect the rights of all the citizens".
"We want to send a signal that diversity makes our society stronger, that together we can do more", she added.
Holding rainbow flags, balloons and a banner reading "For change", participants gathered in Belgrade before marching through the capital.
They will march under the eye of 2,000 police officers - less than half the number deployed for last year's peaceful parade, said organiser Goran Miletic.
While the surprise move made worldwide headlines, sceptics suspected an attempt at "pinkwashing", with Brnabic used as a puppet by Vucic to improve Serbia's image as it campaigns to join the European Union.
Parts of the city Pride route were cordoned off by riot police to prevent clashes between right-wing groups protesting the event.
Serbia is a candidate to join the European Union, but the EU has said it needs to improve minority rights - including for lesbian, gay, bisexual and transgender people.
Serbia's gay community has endured widespread harassment and violence from extremists.
Labor Minister Zoran Djordjevic, Minister of State Administration Branko Ruzic, and Belgrade Mayor Sinisa Mali are also participating in the September 17 parade - the final event of the Pride Week.
A year later, in 2010, marchers walked unhindered along a few city blocks but more than 100 people were injured along the perimeter during an hours-long clash between police and thousands of right-wing extremists and soccer hooligans.
Almost a decade later in 2010, another parade was allowed to go ahead but again descended into violence between anti-gay protesters and police, prompting a three-year ban on the event. Brnabic attended the event past year when she was public administration minister.