Vladimir Putin said he knows who and where they are, but that there was "nothing criminal" about their actions.
British prosecutors have charged the two Russian men with the nerve agent poisoning of ex-spy Sergei Skripal and his daughter Yulia in the English city of Salisbury.
British prosecutors issued a warrant for the suspects' arrest last week.
Britain has said the two suspects were Russian military intelligence officers nearly certainly following orders from the Kremlin, which has denied any involvement in the incidents.More news: State Health Officials Announce First West Nile Deaths In 2018
'It was nearly certainly also approved outside the GRU at a senior level of the Russian state'. We'll see soon. They are civilians of course.
Yulia and Sergei were discovered slumped on a bench in a critical condition - with the nerve agent also poisoning courageous policemen Sgt Nick Bailey, and later residents Charlie Rowley and Dawn Sturgess, who died. "I would like to call on them so that they can hear us today", he said Wednesday.
Mr Skripal and Yulia were poisoned in Salisbury, in the United Kingdom, in March.
His hint that the men could soon break their cover recalls memories of the assassination of former KGB agent Alexander Litvinenko on British soil, when killers Andrey Lugovoy and Dmitry Kovtun publicly refuted the allegations from Russian Federation.
The statement by Putin marked an abrupt shift from Russia's earlier position on the poisoning case that has damaged relations between Moscow and the West. I hope they will come out themselves and speak about themselves.More news: New iPhone XC pictures surface along with preorder and shipment dates
Reacting to Mr Putin's assertion that the spies were civilians, Theresa May's spokesman said Russian Federation has continually replied to requests for an account of what happened in Salisbury with "obfuscation and lies" and he could see "nothing to suggest that has changed".
Calling the poisoning a "sickening and despicable" attack, Javid said it was "unequivocally, crystal-clear this was the act of the Russian state - two Russian nationals sent to Britain with the sole objective of carrying out a reckless assassination attempt".
The Skripals survived the poisoning but a local man, Charlie Rowley, picked up a fake perfume bottle containing Novichok weeks later.
"The president has not communicated with them", Peskov said.
Ms Sturgess died in hospital in July, just over a week after the pair fell ill.More news: Carolinas brace for the worst as monstrous Hurricane Florence nears