The four topless women, who had "In Gay We Trust" scrawled on their bodies, staged their protest by the giant Christmas tree in the centre of the square and were briefly detained by Vatican police.
Tens of thousands are set to march in France later today to denounce government plans to legalise gay marriage and adoption which have angered many Catholics and Muslims, France's two main faiths.
A small protest against the plans is also due to be held in Rome outside the French embassy.
The Femen women's power group has been making headlines since 2010 for topless feminist, pro-democracy and anti-corruption protests in Russia, Ukraine and London.
In September, they set up their first ever "training centre" in Paris.