A Syrian businessman living in Jordan was the first person to pass through the Nassib border post between Jordan and Syria when traffic resumed.
Syrian government forces recaptured the crossing from rebel fighters in July.
UN vehicles were also seen crossing into the Israel-occupied Golan near the Syrian town of Quneitra on Monday.
The news came as Syrian Foreign Minister Walid Muallem discussed "speeding up" the reopening of the country's border crossings with Iraq at a meeting with his Iraqi counterpart Ibrahim al-Jaafari in Damascus.
The first car to cross from Jordan into Syria when the Nassib crossing reopened at 08:00 (05:00 GMT) on Monday was a black Mercedes Jeep.
Jordanian taxi driver Imad Sariheen expressed hope that the resumption of cross-border trade would help ease the "economic hardships" he had suffered.
The closure of Nassib in 2015, when it was overrun by Syrian rebel factions, cut a crucial transit route for billions of dollars of goods and had serious economic repercussions across the region.
The BBC's Martin Patience in Beirut says the reopening of the crossing will bolster the Syrian economy, which has been devastated by a seven-year civil war, and will be seen as a sign of normalisation - that Arab countries are once again willing to deal with Damascus.
Jordan supported the rebellion against Syrian President Bashar al-Assad. But he now appears close to victory, months after the Syrian army defeated opposition fighters in the southern provinces of Deraa and Quneitra with the help of Russian air strikes and Iranian-backed militias.
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