The Tanf crossing in southeastern Syria links the Homs province, including the IS-held ancient city of Palmyra, to Iraq's Anbar province, where IS has a large presence.
The extremist group uses border crossings to shift fighters and resources to different fronts as it seeks to defend and expand its self-styled Islamic caliphate.
IS group captured the Tanf crossing from Syrian government forces in May 2015.
The Britain-based Syrian Observatory for Human Rights, which relies on activists inside Syria, said IS fighters recaptured the crossing, but the Homs Media Center, another opposition outlet, said clashes were still underway.
The Observatory said the FSA fighters crossed into Syria from Jordan to launch the attack, and were supported by airstrikes from the US-led coalition.
The IS-affiliated Aamaq News Agency denied the crossing was ever captured.
IS fighters meanwhile attacked the government's supply route to the contested northern city of Aleppo, killing 15 soldiers, the Observatory said. Government forces repelled the attack and secured the road, according to the Observatory and SANA, the Syrian state news agency.
A US counterterrorism official said the IS group has lost important ground in Syria in recent weeks.
"The overall square kilometers that Daesh has now lost in Syria has increased exponentially in just over the last couple weeks. But it's not just the territories, it's the strategic nature of the territory," said Brett McGurk, President Barack Obama's envoy to the coalition, at a press conference in Baghdad, referring to the extremist group by its Arabic acronym.
The group still controls large swaths of Syria and Iraq, including Iraq's second largest city, Mosul. It has also carried out a number of large bombings in Iraq over the past week that have killed dozens of people.
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