Mr Putin challenged Washington to present the evidence behind its claims to the United Nations Security Council.
US President Barack Obama has said he is considering military action against Syria based on intelligence reports.
The Russia leader’s remarks came after UN chemical weapons inspectors ended their visit to Syria.
They crossed into neighboring Lebanon after four days of inspections, including investigations of what happened in the Damascus suburbs on 21 August.
Hundreds of people including children were killed in the suspected chemical weapons attack, which the US says was carried out by the Syrian government.
Syria said the US claim was “full of lies”, blaming rebels for the attacks.