The Russian Defence Ministry says neither neither Russian nor Syrian war planes had struck a humanitarian convoy near Aleppo the previous day, saying the convoy's whereabouts had only been known to militants on the ground.
The Syrian Red Crescent said the head of one of its local offices and "around 20 civilians" were killed in the attack.
The US State Department said it was "outraged" by the bombing and would be raising the matter directly with Russia.
Igor Konashenkov, a spokesman for the Russian military, denied any Russian involvement in the attack in a statement read out on the state-run Rossiya 24 TV channel on Tuesday however.
"All information on the whereabouts of the convoy was available only to the militants controlling these areas," Konashenkov said.
That appeared to contradict a statement by UN humanitarian aid spokesman Jens Laerke, who told reporters in Geneva earlier on Tuesday, that Russia had been notified about the convoy in advance.
Kremlin spokesman Dmitry Peskov earlier on Tuesday made a series of gloomy comments on Syria, saying the situation was "a source of great concern" and that there was little hope for a renewal of a ceasefire there.
The United Nations has suspended all aid shipments into Syria after the deadly attack on a convoy carrying humanitarian supplies.
The strike on the aid convoy appeared to deliver a death blow to the ceasefire, the latest failed attempt to halt a war, now in its sixth year, which has killed hundreds of thousands of people and created the world's worst refugee crisis.
Syria's army declared the ceasefire over on Monday, hours before the strike.
While the United States initially said it was still hopeful of extending the truce, US officials acknowledged in the wake of the attack that there might no longer be any agreement left to salvage.
That would most likely wreck the last hope of any breakthrough on Syria before the administration of President Barack Obama leaves office in January, meaning his successor will inherit a war that has split the Middle East on sectarian lines and drawn in global and regional powers.
The ceasefire was meant to halt all fighting and allow aid to reach besieged areas, at a time when pro-government forces, with Russian and Iranian military support, are in their strongest positions for years and civilians in many rebel-held are completely cut off from food and medical supplies.
The attack on the convoy of the Syrian Arab Red Crescent destroyed 18 of 31 trucks.
"If this callous attack is found to be a deliberate targeting of humanitarians, it would amount to a war crime," UN aid chief Stephen O'Brien said in a statement. Peter Maurer, president of the International Committee of the Red Cross (ICRC) called it a "flagrant violation of international humanitarian law".
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