"I see that some of our colleagues are pushing the theory that Mr Skripal was almost some kind of human rights activist," Mr Putin said on Wednesday.
The Russian leader, a former intelligence officer himself, then described Mr Skripal as a "traitor to the motherland".
"He's simply a scumbag, that's all."
Putin goes on the attack
By Sarah Rainsford, BBC Moscow correspondent
The verbal attack on Sergei Skripal underlines Vladimir Putin's personal hatred of betrayal. He's said before it's the one thing he can't forgive.
Many Russians share that contempt, and there was applause from some in the conference hall for the comments. But Mr Putin's outburst also seems meant to distract from increasing talk about the two key suspects in the poisoning.
Last month, at another conference, he was asked about the men. He vouched for them personally, saying there was "nothing special" and "nothing criminal" about them.
Crucially, he also insisted they were civilians. Since then, credible evidence has emerged that the men are in fact military intelligence officers and investigative journalists are still digging. On that, Vladimir Putin has so far remained silent.
Who poisoned Mr Skripal?
The UK says two Russian nationals who travelled under the names of Alexander Petrov and Ruslan Boshirov tried to kill the ex-spy and his daughter Yulia.