Russia has been told it must choose between aligning itself with the US and like-minded countries or embracing Syrian President Bashar Assad, Iran and militant group Hezbollah.
The ultimatum came from US secretary of state Rex Tillerson as he embarked on a trip to Moscow following urgent meetings in Italy with top diplomats.
Ahead of the talks in Russia, Turkey's health minister said test results conducted on victims of the chemical attack in Syria confirmed that sarin gas was used.
Recep Akdag said that blood and urine samples taken from the victims confirmed that they were subjected to the nerve agent.
Turkey last week conducted autopsies on three victims of the gas attack who were brought from Syria.
Officials from the World Health Organisation and the Organisation for the Prohibition of Chemical Weapons participated in the autopsies.
Mr Tillerson said it was unclear whether Russia had failed to take seriously its obligation to rid Syria of chemical weapons or had merely been incompetent, but he said the distinction "doesn't much matter to the dead".
"We cannot let this happen again," the secretary of state said.
"We want to relieve the suffering of the Syrian people. Russia can be a part of that future and play an important role," Mr Tillerson added. "Or Russia can maintain its alliance with this group, which we believe is not going to serve Russia's interests longer term."
Mr Putin later said Washington's accusations against the Syrian government over the chemical attack resemble the claims made before the US invaded Iraq in 2003.
Mr Putin, speaking after talks in Moscow with Italian President Sergio Mattarella, said the US invaded Iraq based on false allegations that it had chemical weapons.
He said the US missile strikes on a Syrian air base following accusations that Syria's government used chemical weapons that killed dozens of people last week "strongly resemble the developments of 2003".
Mr Putin added that some in the West are using Syria to cast Russia as a "common enemy".
Russia has argued that civilians in Khan Sheikhoun were exposed to toxic agents from a militants' arsenal hit by a Syrian air strike.
Mr Putin said militants are preparing more "provocations" to blame Damascus.
The Russian President also said they will appeal to the United Nations to investigate the chemical attack in Syria.
Moscow has dismissed suggestions that the Syrian government that it backs could be behind the attack in Idlib province.
Mr Putin told reporters on Tuesday that Russia would appeal to a UN agency in the Hague, urging it to hold an official probe.
Mr Putin also said Russia has received intelligence about planned "provocations" using chemical weapons that would put the blame on the Syrian government.
Since the US launched air strikes against Assad's forces in retaliation for a chemical attack on civilians last week, Trump administration officials have offered mixed messages about whether Washington believes Assad definitely must surrender power - and when.
Mr Tillerson said it was clear the US saw no role for Assad in Syria's future, given that he had lost legitimacy.
"It is clear to all of us that the reign of the Assad family is coming to an end," he said. "But the question of how that ends and the transition itself could be very important in our view to the durability, the stability inside of a unified Syria."
"That's why we are not presupposing how that occurs," Mr Tillerson added.
He said the ceasefire talks that Russia and Iran have helped broker in the Kazakh capital, Astana, could generate momentum towards broader talks about a political transition - if the Astana talks succeed in creating a durable ceasefire.
The resulting political talks would take place under the auspices of the United Nations process in Geneva.
"To date, Astana has not achieved much progress," Mr Tillerson said.
Mr Tillerson spoke after a meeting of the "like-minded" countries was hastily arranged on the sidelines of the summit of the Group of Seven industrialised economies in Italy, days after the US for the first time launched air strikes against Assad's forces.
A key focus since the chemical attack has been on increasing pressure on Russia, Assad's strongest ally, which has used its own military to keep Assad in power.
The US and others have said that Russia bears responsibility for the deaths of civilians at the hands of Assad given Moscow's role in guaranteeing the 2013 deal in which Assad was supposed to have given up his chemical weapons arsenal.
The US raised the stakes significantly on Monday when a senior US official said Washington has made a preliminary conclusion that Russia knew in advance of Syria's chemical weapons attack.
Yet the US has no proof of Moscow's involvement, said the official.
That accusation will hang over Mr Tillerson's visit to Moscow, where he plans with meet with Russia's foreign minister, Sergey Lavrov, and possibly Russian President Vladimir Putin.
The Kremlin declined to say whether Mr Putin would meet Mr Tillerson, in line with its usual practice of not announcing such meetings ahead of time.
The United States has sought to minimise expectations for the trip or the likelihood that the US will leave with any concessions from Russia regarding its support for Assad.
Instead, the US is hoping to use the visit - the first by a Trump cabinet official to Russia - to convey its expectations to Moscow and then allow the Russians a period of time to respond.
Though intended to punish Assad for a chemical weapons attack, the US strikes last week served to refocus the world's attention on the bloody war in Syria, now in its seventh year.
Diplomats gathered in Italy as US officials in Washington floated the possibility of new sanctions on the Syrian and Russian military, plus the threat of additional US military action if Assad's government continues attacking civilians.
At Tuesday's meeting in the walled Tuscan city of Lucca, the G7 countries were joined by diplomats from Muslim-majority nations including Jordan, Qatar, Saudi Arabia, Turkey and the United Arab Emirates.
The inclusion of those countries is important because the US strategy for Syria involves enlisting help from Middle East nations to ensure security and stability in Syria after the Islamic State group is vanquished.
Russia's foreign ministry said in a statement that it hoped for "productive talks".
It said the outcome of the discussions is important not only for the Russia-US relationship, but "for the overall atmosphere on the world stage".