Coalition warplanes targeted a convoy of Syrian government forces and Iranian-backed militia close to the Iraq border on Thursday.
The action was to protect British and American special forces based in al Tanf, southeast Syria.
“It is illegitimate, unlawful and yet another gross violation of Syria’s sovereignty,” Russian foreign minister Sergei Lavrov said in televised comments while on a trip to Cyprus.
He also said Moscow was “concerned” that the “emerging understanding” of the need to unite efforts against Islamic State and the former Nusra front was “starting to erode”.
Russia is a key ally of Syrian dictator Bashar al Assad’s regime, which called the US-led action a “brazen attack” and insisted it would “not be intimidated” by the assault.
The Syrian regime claimed the bombardment killed a number of people and said it revealed the “falseness” of the coalition’s claim to be solely fighting IS in the war-torn country.
“The Syrian Arab Army is fighting terrorism on its territory, and no party has the right to determine the course of its operations,” a source told AFP.
It was the first close confrontation between the US military and forces supporting Mr Assad.
US defence secretary General James Mattis denied the destruction of the convoy, which included a number of tanks, marked an escalation of America’s intervention in Syria.
He said: “We are not increasing our role in the Syrian civil war. But we will defend our troops.”
In a statement on Twitter, Operation Inherent Resolve said the Russians attempted to get the convoy to turn back.
“Coalition struck Syrian pro-regime forces advancing in a de-confliction zone near al Tanf posing a threat to US partner forces,” it said.
“This was despite Russian attempts to dissuade pro-regime movement towards al Tanf, coalition aircraft show of force and warning shots.”
US and UK special forces have been training Syrian rebels at a heavily fortified base in al Tanf.
A 34-mile defensive zone exists around the base, and it is believed these forces strayed inside that.
“They were potentially probing to see how close they could get to al Tanf,” an anonymous US official said.
The convoy was believed to have been about 17 miles away from the base when it was hit.
The claim that Russia was warned and was still unable to stop the convoy will call into question how much influence their commanders have over Assad and Iranian forces in parts of Syria.