The US forces launched strikes on Wednesday, targeting a weapons storage facility in eastern Syria.
The Pentagon said that the Iranian Revolutionary Guard and its affiliated groups were using it, which is the second strike launched by the country within weeks.
Nine were killed in a US air strike that targeted a military facility linked to Iran on Wednesday in the city of Deir al Zour in eastern Syria.
As tensions escalate in light of the ongoing conflict between Israel and Hamas, US and coalition forces in Iraq and Syria have been attacked at least 40 times by Iranian-backed factions since the beginning of October.
Around 45 US traumatic brain injuries or minor wounds.
Defense Secretary Lloyd Austin said in a statement that the strikes were carried out by two F15 jets and came in response to recent attacks targeting US forces.
Austin added that attacks on US forces must stop, and continued, “If Iranian proxies continue to attack US forces, we won’t hesitate to take further necessary measures to protect our soldiers”.
The United States has occasionally carried out retaliatory strikes on Iranian-backed forces in the region after they attacked US forces.
On October 26, US forces attacked two facilities used by the Iranian Revolutionary Guard and the groups it supports.
The United States has 900 soldiers in Syria and 2,500 soldiers in Iraq on a mission to provide advice and assistance to local forces trying to prevent the return of ISIS, which in 2014 took control of large areas of the two countries’ territories but was later defeated.
There is growing concern about the expansion of the conflict between Israel and Hamas in the Middle East and the transformation of American forces stationed in various bases into targets.
Earlier Wednesday, the Iran-aligned Houthi group shot down an US MQ-9 drone near Yemen.
The United States has sent warships and combat aircraft, as well as two aircraft carriers, to the region since the outbreak of the conflict between Israel and Hamas on October 7, in an attempt to deter Iran and the factions it supports.
The number of forces recently sent to the region is estimated at thousands.
The US military is taking new steps to protect its forces in the Middle East with the increase in attacks suspected of being launched by Iranian-backed groups, and leaves the door open to the possibility of evacuating military families if necessary.
Officials said the measures include increasing US military patrols, restricting access to base facilities housing troops, and increasing intelligence gathering, including using drones and other surveillance operations.