"Berlusconi is on trial for living with women, but if he were a homosexual, nobody would dare touch him," Putin said during a discussion with
international experts and journalists at the Valdai Club in the northwestern Novgorod region.
The remark was met by roars of laughter from the audience.
Berlusconi, 76, is appealing a conviction for having sex with an underage prostitute nicknamed Ruby the Heart Stealer.
The Valdai forum is a discussion organised annually by Russia's state news agency RIA Novosti that invites various foreign speakers to discuss Russia.
Putin has come under fire for his anti-gay stance since he signed a controversial "gay propaganda" law in June that bans disseminating information
about homosexuality to minors.
Outrage in the West has also led to calls to boycott the Winter Olympics in Russia's Sochi resort next year.
Putin frequently defends "traditional" values and said at the Valdai club that Western countries had forgotten their Christian roots and were "denying
moral principles and traditional identities," including in "sexual" matters.
The Russian president is known to be friends with Berlusconi, who has flown in to Russia for several of Putin's birthdays and to celebrate his victory in presidential polls last year.
The former Italian prime minister has also been accused of homophobia.
"I am always working flat out and sometimes I look at beautiful women…It's better to be passionate about beautiful women than to be gay."
Berlusconi said in the midst of the sex scandal in 2010.