The Russian leader warned against "ill-considered and unsafe actions ... that would have consequences beyond conjecture".
The UN Security Council was to meet again on Friday, at Russia's request, to try to defuse the standoff, as US President Donald Trump appeared to back away from imminent action, days after warning Russian Federation to "get ready" for missile strikes.
Britain's U.N. Ambassador Karen Pierce, meanwhile, rejected a charge by a Russian defense ministry spokesman that Britain was involved in staging a fake chemical weapons attack in Douma.
British Prime Minister Theresa May and French President Emmanuel Macron said the United Kingdom and France had joined in the attack.
US Defence Secretary Jim Mattis struck a cautious note, telling lawmakers that the need to "stop the murder of innocent people" had to be weighed up against the risk of things "escalating out of control".
But he also appeared anxious to avoid a wider conflict, saying France would "in no way allow an escalation".
The U.S. State Department said the United States has proof at "a very high level of confidence" that the Syrian government carried out the attack but is still working to identify the mix of chemicals used. Striking a conciliatory note, it said: "The President of the Republic called for dialogue with Russian Federation to be maintained and stepped up to bring peace and stability back to Syria".
Inspectors from the Organisation for the Prohibition of Chemical Weapons are expected to arrive in Syria over the weekend to investigate the reported attack, following an invitation from Damascus.
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There was no word from Putin himself, though Russian Foreign Minister Sergei Lavrov said Moscow was in contact with Washington to discuss an atmosphere which he described as alarming.
He went on to say that the alleged chemical attack in Douma is just an American pretext and he blamed the Syrian opposition, not the Assad regime, for it.
But this was balanced by a warning from France's ambassador to the U.N., Francois Delattre, who told the Security Council that the Syrian government's decision to use chemical weapons again meant they had "reached a point of no return".
The Trump administration did not lay the blame only at the feet of Syria's leader.
The capture of Douma from rebels who evacuated this week has clinched a major victory for Assad, crushing what was once a center of the insurgency near Damascus, and underlines his seemingly unassailable position in the war.
"Without the sanction of the UN Security Council, in breach of the UN charter and the norms and principles of worldwide law, an act of aggression was committed against a sovereign state", the Kremlin said.
US allies have offered strong words of support for Washington but no clear military plans have yet emerged.
The Kremlin on Saturday condemned Western air strikes on Syria where its armed forces are backing President Bashar al-Assad.