Speaking to reporters, Defense Secretary Jim Mattis said the U.S. will defend its troops in case of "aggressive" steps against them. He was asked if the airstrike increases the U.S. role in the Syrian war.
"We are not increasing our role in the Syrian civil war, but we will defend our troops," Mattis said.
The "defensive" strike was also an apparent signal to Assad to keep his forces out of a zone where U.S.-backed rebels are fighting the Islamic State (ISIS).
"This action was taken after apparent Russian attempts to dissuade Syrian pro-regime movement south ... were unsuccessful, a coalition aircraft show of force and the firing of warning shots," the U.S.-led coalitions aid. It said coalition forces have been operating in the area "for many months training and advising vetted partner forces" in the battle against ISIS.
The U.S. strike marks a new approach in what has become a crowded and complicated war zone. The strike was the coalition's first on pro-Assad forces in the battlefield. The coalition had so far kept its military operations focused on ISIS militants and al-Qaida-linked groups.
Last month, the U.S. fired 59 missiles at a government air base in central Syria as punishment for a chemical attack blamed on Assad's forces that killed nearly 90 people.
The U.S. is backing Syrian Kurdish forces who are also fighting ISIS to the country's east. U.S. troops have sent patrols in the area to act as a buffer between Turkish troops and Kurdish fighters. Turkey views the U.S-backed Kurdish fighters as an extension of its own insurgent group.
In recent days, near the border with Jordan, another set of U.S-backed rebel fighters have been on a collision course with government troops in the area of Tanf.
The government launched a new offensive in recent days in the area. Activists say pro-government militiamen, mainly from Iran and the Lebanese Shiite militant Hezbollah, have deployed there aiming to secure the main highway that runs from Damascus to Baghdad and Tehran.
Tensions have been building as part of a race for control of territory stretching from the provincial capital of Deir el-Zour in northeastern Syria to the Iraq border.
The area gained attention as the battle for the Iraqi city of Mosul escalated in recent weeks. An estimated 10,000 ISIS fighters uprooted from Mosul are believed to be massing in the border area.
A Syrian opposition media group, the Palmyra News Network, said the attack occurred at the Zarka juncture, about 17 miles from the border, destroyed a number of vehicles and caused casualties. The Syrian Observatory for Human Rights also said the strike destroyed vehicles and killed eight militiamen. There was no immediate comment from the pro-government side.
In September 2016, the coalition erroneously struck Syrian government troops in Deir el-Zour, killing more than 90 soldiers. The U.S. at the time said it was a mistake, as it was targeting ISIS positions.