"Berlusconi is being tried because he lives with women. If he were homosexual, no one would lift a finger against him," he told a gathering of journalists and Russia experts.
Putin's comment provoked laughter in the audience over what appeared to be a reference to criticism abroad over a law he signed this year banning the spread of "anti-gay propaganda" among minors in Russia.
Berlusconi is appealing against a conviction for paying for sex with a former nightclub dancer when she was under 18. He has also been convicted of tax fraud.
He and Putin regard each other as friends and the former KGB spy has proved loyal, telling a conference in 2011 that "however much they nag Signor Berlusconi for his special attitude to the beautiful sex ... he has shown himself as a responsible statesman."