The aircraft struck the convoy as it headed toward a remote coalition garrison near the border with Jordan, a United States defense official said.
The pro-regime convoy had come within 18 miles the al-Tanf base, which houses USA and British special forces in southern Syria, and breached the "deconfliction zone" around the base.
Despite the strikes, Pentagon chief Jim Mattis said the United States was "not increasing our role in the Syrian civil war, but we will defend our troops".
US-led anti-ISIS coalition has carried out air strikes against a convoy of a Iran-backed Shiite militia supporting the regime of Syrian President Assad. Last summer, Syrian fighters trained by the USA who operated out of al-Tanf were bombed by Russian jets.
Syrian and Iranian-backed militiamen were reportedly moving towards the Tanf base, which is used by rebel fighters and Western special forces personnel.
Several hundred USA advisers are in Syria to help train and organize a force to defeat ISIS.
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Attempts to halt the convoy included a call to the Russians - who are working with the Syrian regime- the official said, before "show of force" above the vehicles, followed by warning shots. Pro-regime forces had crossed a pre-agreed deconfliction zone and continued to advance despite warning strikes, he added.
It said four vehicles were also destroyed. It was unclear if they resulted in any deaths.
US-led coalition aircraft bombed pro-Syrian regime forces Thursday that "were advancing well inside an established de-confliction zone", according to a statement issued by Operation Inherent Resolve, the official name for the coalition fighting ISIS.
The Syrian military official clarified on Friday that the area attacked was a military position along Tanf highway in the desert.
That thinking changed in April with the missile strike fired against Assad's air force in response to Assad's deadly chemical weapons attack.
Ominously, the Syrian regime has its own base fairly close to the one American forces are using to train opposition fighters, and the Syrian base is said to be ready to support "about a battalion's worth of troops". Last month, the U.S. "And that's been a going-in policy of ours for a long time".
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