Ana Brnabic, 41, became one of the few openly gay government leaders in the world when she came to power in June, but activists say homophobia remains a widespread problem in the conservative Balkan country.
Setting off on the Pride parade with hundreds of activists waving rainbow flags and balloons, Brnabic was keen to give a more positive message, saying Serbia was "finally showing what I believe is its true face".
She encouraged gay youngsters to be "full of understanding for people who are very traditional and who do not yet truly understand what this is about".
In 2010, Belgrade's Pride parade descended into clashes between anti-gay protesters and police, injuring more than 100 people and prompting a three-year ban on the event.
This is the fourth consecutive year the march has gone ahead under a huge security presence, and the first time a prime minister has attended.
Around 2,000 armed officers guarded the cordoned-off streets as activists marched through the city, accompanied by a soundtrack of pop music and a police helicopter buzzing overhead.
Brnabic was chosen for the top job by her predecessor, Aleksandar Vucic, after he was elected president. She had entered politics less than a year earlier as public administration minister.
Her appointment, endorsed by parliament, made international headlines. But 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.
Brnabic, a technocrat with a business background, has firmly dismissed allegations that she was chosen merely because of her sexual orientation.
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