The singer paused during a Euro 2012 concert to voice his disappointment following reports of physical attacks on members of the Ukrainian gay community.
John, who was playing as part of an AIDS charity event in Kiev yesterday (June 30), described the country as his "second home" following his failed adoption of an HIV positive baby named Lev from a Ukrainian orphanage in 2009.
"Recently I read about violence against gay people in Ukraine," he explained. "Beating up gay people is wrong. This for me does not symbolise Ukraine.
"I plead with you. Stop the violence against gay people."
Euro 2012 concludes in Kiev today (July 1). Prior to the opening match of the tournament on June 8, a gay pride march in the capital was cancelled and one of its leaders was violently assaulted.
Last Saturday (June 22), a leading Ukrainian gay rights activist revealed that he had also received hospital treatment in Kiev after being beaten by a group of people who screamed homophobic insults at him.
John has been in a civil partnership with David Furnish since 2005 and frequently speaks out about gay rights. He is also head of the Elton John AIDS Foundation, a charity that supports the fight against HIV across the globe.
Watch a video of Elton John singing 'Your Song' at the Queen's Diamond Jubilee celebrations below: