Pentagon spokesman Navy Captain Jeff Davis said the Iran-made drone had "dirty wings," meaning it was armed.
The US military said it had shot down an armed, Iranian-made drone that had been bearing down on its forces near a garrison in Syria's south-east on Tuesday, in the latest sign of increasingly frequent confrontation with Damascus and its allies.
The incident closely followed Sunday's US downing of a piloted Syrian army jet in the southern Raqqa countryside after it dropped bombs near US-backed forces.
The US-led coalition said the location was close to where another pro-regime drone, which intelligence sources had also identified as Iranian, was shot down on June 8 after dropping bombs near coalition forces.
In an indirect reference to Iranian-backed forces that have been gathering in the eastern desert region, a US-led coalition statement cited a recent escalation of tensions and said it would not "tolerate any hostile intent and action of pro-regime forces."
In the latest incident, the Pentagon said a US F-15 aircraft flying over Syrian territory fired on the drone after it displayed hostile intent and advanced on coalition forces.
Pentagon spokesman Navy Captain Jeff Davis said it had "dirty wings," meaning it was armed.
"I can tell you it was an Iranian-made drone," Davis said, declining to speculate on who specifically was operating it.
A Western intelligence source said it was "unquestionably Iranian."
"They are testing the limits," the source told Reuters without elaborating.
The area falls in a part of Syria that was recently identified as a military priority by Damascus, and is seen as strategically important for Iran as it seeks to secure a land corridor between forces it backs in Syria and Iraq.
Russia, like Iran, an ally of Syrian regime leader Bashar al Assad, issued a warning of its own to the United States in response to the downing of the Syrian jet, saying on Monday it would view as targets any planes flying west of the Euphrates river, though it stopped short of saying it would shoot any down.
Still, the Pentagon said it had not seen hostile action by Moscow.
"Public statements aside, we have not seen the Russians do any actions that cause us concern. We continue to operate, making some adjustments for prudent measures," Davis said.
Meanwhile, the US and Russian militaries swapped accusations about an unsafe intercept involving a US spy plane and a Russian fighter jet over the Baltic Sea.
On several occasions in recent weeks, warplanes of the US-led coalition have also struck pro-regime forces to prevent them advancing from the Al Tanf garrison in southeastern Syria at a spot where the country's borders join Iraq and Jordan.
Washington also described those strikes as self-defence.
Conflict continues on the Jordanian border
Regime air and artillery bombardments hit rebel-held areas of the Syrian city of Daraa, on the border with Jordan, on Tuesday after a two-day ceasefire expired, witnesses, rebels and the army said.
Syrian state television, quoting army sources, said they had resumed the offensive, which took place as US and Russian officials were holding another round of secret talks on creating a "de-escalation zone" in southwestern Syria that would include Daraa.
US and Russian officials had agreed to a ceasefire, which ended on Monday, during talks in Amman aimed at strengthening goodwill before more detailed negotiations on setting up the "de-escalation zone," diplomats in Jordan said.
On Saturday, the Syrian army said it would suspend combat operations in Daraa for 48 hours to support "reconciliation efforts."