A Pentagon spokesman told reporters the US's aim in Syria and Iraq was to "fight ISIS, and fight ISIS only".
One rebel group has left a joint base after clashing with the regime.
It comes after President Donald Trump ended the CIA's clandestine programme to provide weapons and supplies to Syrian rebel groups.
That decision to end the programme, which started under the Obama administration in 2013 in an attempt to put pressure on President Bashar al-Assad to stand down, was made more than a month ago as part of an effort to improve ties with Russia, officials said last week.
Russia's military support has helped Mr Assad hold on to power during the six-year civil war that has left more than 300,000 people dead and displaced 11 million others.
Whether this latest announcement is related is unclear but on Thursday, a spokesman confirmed a group known as Shohada Al Quartyan, which was part of the US-led coalition in southern Syria, had split off after being told to focus on IS and not fighting against Mr Assad's government forces.
Coalition spokesman Colonel Ryan Dillon said they "unilaterally, without US or coalition permission or coordination" conducted patrols outside a specified zone and engaged in "activities not focused on fighting ISIS".
According to CNN, the group was part of the "vetted Syrian forces" - made up of opposition fighters - being trained and equipped by the coalition.
However, Col Dillon told reporters in Washington: "We have made it very clear time and again our goal in Syria and Iraq is to fight ISIS and fight ISIS only [and] we've asked [our partner forces] to be committed to that same mission.
"We had a partner force we were working with who wanted to pursue other objectives, and those objectives were not consistent with defeating ISIS. So we have since talked with them and made them know that we cannot support them if they want to pursue objectives other than defeating ISIS.
"We are no longer going to support this particular group because that is what they want to do."
He said they would try to get back the equipment the group, which operates in the Al-Tanf area, near the Jordanian border, was given.
The US has warned its coalition partners in Syria to focus exclusively on the fight against so-called Islamic State (IS) and not regime forces.