The attack destroyed "multiple facilities located at a border control point used by a number of Iranian-backed militant groups", it said.
President Joe Biden approved the action in response to recent attacks against US and coalition personnel in Iraq.
A civilian contractor was killed in a rocket attack on US targets in Iraq earlier this month.
A US service member and five other contractors were injured when the rockets hit sites in Irbil, including a base used by the US-led coalition.
Rockets have also struck US bases in Baghdad, including the Green Zone which houses the US embassy and other diplomatic missions.
After Trump, what will Biden do about Iran? The Pentagon named Kataib Hezbollah and Kataib Sayyid al-Shuhada as two of the Iran-backed militias it targeted in Thursday's strike in eastern Syria.
It called the action a "proportionate military response" that was taken "together with diplomatic measures", including consulting coalition partners.
"The operation sends an unambiguous message," the Pentagon statement said.
"President Biden will act to protect American and Coalition personnel. At the same time, we have acted in a deliberate manner that aims to de-escalate the overall situation in both eastern Syria and Iraq."
The Syrian Observatory for Human Rights said the strikes had killed at least 17 pro-Iranian fighters.
"The strikes destroyed three lorries carrying munitions," the observatory's Rami Abdul Rahman told AFP. "There were many casualties."
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