A pro-Assad convoy, including tanks, apparently ignores warnings from the US and Russia before it is destroyed.
The airstrikes, which killed Syrian government forces and Iranian-backed militia, occurred close to the border with Iraq.
US defence secretary Jim Mattis denied the destruction of the convoy, which included a number of tanks, marked an escalation in the American role in Syria.
"We are not increasing our role in the Syrian civil war," he said. "But we will defend our troops."
In a statement on Twitter, Operation Inherent Resolve said: "Coalition struck Syrian pro-regime forces advancing in a de-confliction zone near al Tanf posing a threat to US partner forces.
"This was despite Russian attempts to dissuade pro-regime movement towards al Tanf, coalition aircraft show of force and warning shots.
"Coalition forces have operated in the al Tanf area for many months training and advising vetted partner forces who are fighting ISIS."
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.
US and UK special forces have been training Syrian rebels at a heavily fortified base in al Tanf.
Islamic State attacked the base last month and recently regime forces have been pushing forward nearby, raising fears of a collision with coalition forces.
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 At Tanf," an anonymous US official said.
The convoy was believed to have been about 17 miles away from the base when it was hit.
Iranian troops are said to be commanding the pro-Assad forces and a number of Lebanese Hezbollah fighters.
Exact casualties figures have not been provided, although some reports estimate 50 were killed.
Al Tanf is part of a region known as the Badia, a vast, sparsely populated desert area that stretches to the Jordanian and Iraqi borders.
US-backed rebels have secured larges areas of the Badia from Islamic State in recent months, but the Syrian army and Iranian-backed militia have moved hundreds of troops with tanks to the town of Sabaa Biyar, near the strategic Damascus-Baghdad highway.