Police and prosecutors last week said Alexander Petrov and Ruslan Boshirov had been identified as members of the GRU, Russia’s military intelligence service.
Russia has repeatedly contested the allegations and on Wednesday Putin escalated the war of words by denying the men were members of military intelligence.
In an address to the Easter Economic Forum in Vladivostok, he said: ‘Of course, we looked who these people are. We know who they are, we have found them already.’
He added: ‘There is nothing special and nothing criminal about it, I’m telling you.’
Questioned on whether the pair were civilians, he replied: ‘Of course they are civilians.’
In an unusual move, he then called on Petrov and Boshirov to appear before the media to talk about ‘themselves’.
His intervention risks widening the gulf between Russia and the UK over the attempted assassination, which triggered a wave of diplomatic expulsions by both sides.
Former GRU officer Sergei Skripal and his daughter Yulia were left critically ill after being exposed to the military grade nerve agent Novichok in Salisbury in March.
Detectives believe it is likely the two suspects, thought to be aged around 40, travelled under aliases and that Petrov and Boshirov are not their real names.
Prosecutors deem it futile to apply to Russia for the extradition of the two men but a European Arrest Warrant has been obtained and the authorities are also seeking the assistance of Interpol.
Officers have formally linked the attack on the Skripals to events in nearby Amesbury when Dawn Sturgess, 44, and her partner Charlie Rowley, 45, were exposed to the same nerve agent.
Ms Sturgess died in hospital in July, just over a week after the pair fell ill.
A police officer who visited the home of the Skripals shortly after the attack, Nick Bailey, was also left critically ill from exposure to the substance.