The airstrikes are understood to have hit a pro-regime militia that was advancing on US and coalition forces at At Tanf, according to Operation Inherent Resolve, the official name for the coalition fighting ISIS.
A DoD official told Voice of America that the strikes were "not a change in policy but [an] instance of [a] commander on the ground's decision for force protection".
The United States military has carried out an airstrike against pro-Assad forces in Syria.
The forces came within a 34-mile defensive zone around the at-Tanf base in southern Syria, according to the command.
The Americans first alerted their Russian colleagues using a special hotline the two sides set up to ensure their aircraft don't operate in the same airspace.
"A convoy going down the road didn't respond to numerous ways for it to be warned off from getting too close to coalition forces in At-Tanf", the official told AFP on condition of anonymity.
One opposition media group, the Palmyra News Network, says the attack at the Zarka juncture, about 27 kilometres from the border, destroyed a number of vehicles and led to casualties.
Riyadh has been pushing for a reset of bilateral relations, which winds back a U.S. pivot towards Iran under the Obama administration in favour of renewed ties between Washington and Riyadh that had been deeply strained during the former president's second term. Thursday's incident involved two breaches, according to The New York Times, which reported that F-22 fighters were dispatched to scare off a Syrian SU-22 fighter-bomber.
The government launched a new offensive in recent days in the area, and activists say pro-government militiamen, mainly from Iran and the Lebanese Shiite militant Hezbollah group, have deployed there aiming to secure the main highway that runs from Damascus to Baghdad and beyond, to Tehran.More news: Trump's Latest Political Mess Helps Push Gold Prices Higher
The area has been a source of tensions as both government forces and US -backed rebels advance there.
The US-backed Maghaweer al-Thawra rebel faction said the convoy was 27km (17 miles) away and contained four tanks.
The American strikes were the first against Assad positions since the Pentagon rained 57 Tomahawk missiles on the Shayrat air base near Homs. No Syrian or Russian troops were killed.
The airstrike occurred after a convoy of pro-Assad militia forces appeared to be headed toward at-Tanf, a town located near Syria's southeastern border with Iraq, according to the US official.
The Syrian regime and Iran probably calculated that the USA would not come to the aid of its rebel allies in al-Tanf, as it untraditionally did on Wednesday. The U.S. -led coalition operates a training facility there for Syrian opposition forces fighting ISIS. After the initial assault was pushed back, coalition jets destroyed the remaining ISIS fighters.
At the same time, the Assad regime has also become more publicly critical of Jordan, a key Arab member of the anti-ISIS coalition, owing to reports that the Jordanian army was mobilizing near the Syrian border.
And in June previous year, Russian warplanes bombed the outpost, reportedly just one day after British special forces had pulled out.
These latest strikes are yet another example of the weirdness of the US position in Syria.