Washington's lead diplomat said the US was "quite concerned" by a recent confrontation that saw ally Israel bomb what it described as Iranian targets in Syria.
"This again illustrates why Iran's presence in Syria is only destabilising to the region," Tillerson said at a press conference with his Jordanian counterpart.
"We think Iran needs to withdraw its military, its militia from Syria, and allow the hope for peace process to take hold."
US foe Iran and regional militias it backs, including Lebanon's Hezbollah, have played a key role in propping up leader Bashar al-Assad, heightening fears over Tehran's spreading influence.
Tillerson -- who is on a tour of the Middle East -- held closed-door talks with Syrian opposition negotiators in Amman in the wake of a January peace summit hosted by regime backer Russia.
Tillerson said there were "important milestones" achieved at the conference in Russia's Black Sea resort of Sochi, most importantly a "unified commitment" to the UN efforts.