The Pentagon confirmed that an air strike was carried out Friday in support of the New Syria Force, a US-allied group.
"We'll take action to defend the New Syria Force that we've trained and equipped," Pentagon spokesperson Commander Bill Urban told AFP.
Read: US, Turkey join forces to drive Islamic State from Syria
He said "last Friday was the first one," referring to the air strike.
Earlier, a senior administration official said the United States had hit al Qaeda's Syrian affiliate the al Nusra Front in response to attack on US trained rebels.
President Barack Obama's administration said Monday it was prepared to take "additional steps" to defend US-trained and equipped forces, warning Bashar al Assad's regime "not to interfere."
"The president approved this recently upon the recommendation of his senior military advisers," a senior administration official told AFP.
White House spokesmperson Josh Earnest said Assad's regime had not so far hampered US-backed forces, but he nonetheless raised the possibility of strikes against it should the need arise.
The United States, Earnest said, was "committed to using military force where necessary to protect the coalition-trained and equipped Syrian opposition fighters."
The decision was taken under a 2001 rule authorizing the use of military force against terror groups, which critics say has already been stretched too far.
Officials argue that authority includes the ability to provide "defensive fire support."
The United States has trained and equipped a number of fighters -- screened and determined to be "moderate" -- to operate against the extremist Islamic State organization.
But US-backed forces have yet to play a major role in turning the war and its fledgling local ground force has already suffered a series of reversals.
A 54-strong unit inserted into the rebels' Division 30 has come under withering attack from the al Nusra Front, with several members reportedly killed or captured.
Micah Zenko of the Council on Foreign Relations said the "truly significant decision" could potentially extend well beyond that small force.
US forces are "interspersed among large coherent units of several hundred fighters," he said, explaining that: "You can't give air cover just to individual rebels."
The United States recently agreed with Turkey to create what has been termed an "Islamic State-free zone" in northern Syria.
Details of the zone "remain to be worked out", according to a senior administration official, who asked not to be named.
Read: US-led anti-IS strikes kill family in Syria: monitor
It would, however, entail Turkey, NATO's only mainly Muslim member, supporting US "partners on the ground" already fighting the extremists.
Ankara has also granted the United States permission to use one of its bases to carry out air raids against the group.
Washington has long pushed for the use of the Incirlik base due to its location relatively close to Syria just outside the Turkish city of Adana, but Turkey had hesitated for months.
Monday's announcement comes as diplomatic efforts to halt the carnage in Syria resume.
An estimated 140,000 people have died in the conflict, which began as an uprising against the Assad regime but has morphed into a multipronged religious and ethnic civil war.
A UN envoy recently presented his plan to resuscitate failed talks and foreign ministers from the United States, Russia and Saudi Arabia held talks in Qatar on Monday.
The trio agreed to the "need for a meaningful political transition" according to State Department spokesperson Mark Toner.
But Russian Foreign Minister Sergei Lavrov later condemned Washington's move toward a more robust involvement in Syria.
"We believe it's counterproductive to announce publicly that some US-trained armed groups... will be under the protection of the coalition's air forces," Lavrov said.
Separately on Monday, the US Treasury Department imposed sanctions on seven entities and four individuals its says are providing energy products to Assad's regime, and named seven vessels as blocked property.
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