Several thousand police officers were guarding the procession in the capital and the rally was peaceful, despite far-right groups making threats last week to attack it.
People were marching with rainbow flags and carried placards saying: “Love has no gender.”
Bohdan Hloba, one of the organisers, said: “The road to equality in Ukraine is difficult as well as dangerous.
“We have been threatened with a ‘bloodbath’ but every step of this march gives us hope.”
Authorities sanctioned gay rights marches when the pro-Western government came into power after the 2014 revolution, but earlier gatherings have been small and have come under attack from far-right groups.
Kiev city police cordoned off nine streets and closed one subway station yesterday to ensure tight security and prevent clashes.
However, a few anti-gay activists did get in, although they were not violent. “I’m against gay propaganda that these sick people have organised here in collusion with authorities,” said Serhiy Hashchenko, 56, a father of 12 who went to the march carrying a placard saying “Ukraine is no Sodom”.
Ultra-nationalist radicals who had threatened to disperse the march were watching it from the security perimeter lined with riot police.
Last year, a gay pride march in Kiev was called off less than half an hour after it began as right-wing activists pelted the marchers with smoke grenades.
Metal detector at entrance to gay pride/'equality march' in Kiev #ukraine huge security operation to avoid violence pic.twitter.com/8d6D0aiVU4
— Tom Soufi Burridge (@TSoufiBurridge) June 12, 2016