"They are civilians, of course," Putin said, contradicting the British government's assertion that they are officers of Russia's military intelligence agency, known as the GRU.
He said that there was "nothing criminal" about the two men and that he hoped they would come forward and tell their own story, adding: "That would be better for everybody."
Putin gave no indication that Russia would help Britain pursue the suspects, and his poker-faced remarks appeared to indicate that it would not.
Russia adamantly denies involvement in the poisoning, which had added to severe strains in ties between Russia and the West.
Putin's declaration came seven days after British authorities announced that they had charged two Russian men, identified as Aleksandr Petrov and Ruslan Boshirov, with carrying out the poisioning on March 4.
They accused the pair of smuggling the Soviet-designed nerve agent Novichok into Britain in a fake perfume flask and smearing some of the substance on the front door of Sergei Skripal's home in the English city of Salisbury, where the former GRU officer settled after being sent to the West in a Cold War-style spy swap in 2010.
The attack left Sergei Skripal, 67, and Yulia Skripal, 34, in critical condition, but both have recovered after weeks in the hospital.
A couple who authorities said found the perfume bottle after it was discarded by the attackers fared worse: Charlie Rowley recovered after treatment in the hospital but his partner, Dawn Sturgess, 44, died on July 8.
British authorities have said that a European arrest warrant has been issued for the two Russians, who they suspect were using aliases.
Putin made no comment about whether the names they used were real.
British Home Secretary Sajid Javid said on September 9 that Britain will catch the two men and bring them to prosecution if they ever step out of Russia.
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 purpose of carrying out a reckless assassination attempt."
Javid described the GRU as a "very well-disciplined organization" that would "only act with orders from the highest level of the Russian government."
Britain's security minister, Ben Wallace, said on September 6 that Putin "ultimately" bears responsibility for the poisoning because "it is his government that controls, funds, and directs the military intelligence."
The poisoning led Britain, the United States, the European Union, and others to carry out a series of diplomatic expulsions and financial sanctions against Moscow.
It has further damaged already severely strained relations between Russia and the West and has been a cause for solidarity at a time when Western officials accuse Moscow of seeking to cause rifts in relations between Western countries.