Islamic State had controlled the al-Tanf border crossing, which is also near the Syrian-Jordanian border, since May last year after seizing it from Syrian government forces. It had been the last border crossing with Iraq that was under the control of the Syrian government.
IS, which controls a swathe of territory spreading from Iraq into central Syria, still controls the Bukamal Syria-Iraq border crossing near Deir al Zour.
The Observatory said the rebel fighters who took the crossing crossed into Syria from Jordan.
The crossing is a 240 km (150 miles) drive from Palmyra, also known as Tadmur, which has been under IS control since the middle of last year.