"Why are the Russian military so obsessed with castration?" he asked.
The word "punishment" cropped up some 15 times in Mr Zelensky's speech, and was the first of his five non-negotiable conditions for peace.
Russia must face consequences for its aggression, he said, through further sanctions and by the UN stripping Moscow of its powerful role as a permanent Security Council member.
He also called for Ukrainian lives to be protected, and for the country's internationally-recognised borders to be respected.
As his fourth and fifth conditions, he called for new security guarantees for Kyiv, and for the world to unite in calling out Moscow's armed aggression.
Later on Wednesday, the European Union's foreign policy chief said EU countries had agreed to hit Russia with new sanctions.
Josep Borrell told reporters the new restrictions would target Russian individuals and the country's economic sectors.
Sporting his signature green t-shirt in his video, Mr Zelensky thanked the 101 countries at the UN which voted to allow him to address the assembly in a video rather than in person.
He blasted the seven countries including Russia which voted against his video appearance, and criticised those which have remained neutral during the conflict.
Despite Mr Zelensky's suggestion that Russia was not serious about negotiations, the two nations did complete a major exchange of prisoners of war on Wednesday, BBC reported.
215 fighters were returned to the Ukrainian side, including 10 foreigners - while Russia received 55 soldiers and the pro-Russian politician Viktor Medvechuk, who faced treason charges in Ukraine.
