Britain has charged two Russian men, identified as Alexander Petrov and Ruslan Boshirov, with attempting to murder Skripal and his daughter Yulia by spraying a chemical weapon on Skripal's front door in the southern English city of Salisbury in March.
"The fact is that neither Petrov nor Boshirov have nothing to do with Putin, and the Kremlin of course," Dmitry Peskov was quoted as saying.
Putin said on Wednesday that the two men were civilians and that there was "nothing special and criminal" about them.
The duo appeared on Russia's state-funded RT television station on Thursday, insisting they were innocent tourists who had flown to London for fun and visited the city of Salisbury to see its cathedral.
Britain has said the two men were Russian military intelligence officers almost certainly acting on orders from high up in the Russian state. Russia has vehemently denied any involvement in the incidents.
Skripal - a former colonel in Russian military intelligence who betrayed dozens of agents to Britain's MI6 foreign intelligence service - and his daughter were found slumped unconscious on a bench in Salisbury in March. They spent weeks in hospital before being discharged.
(Reporting by Gabrielle Tétrault-Farber; Editing by Mark Potter and Emelia Sithole-Matarise)