The Southern Front alliance took full control of the 52nd Brigade base in Daraa province after 24 hours of fierce clashes, a spokesman for the group told AFP.
"The 52nd Brigade base was fully liberated from the regime army," Major Essam al-Rayes said, adding that at least 2,000 rebel forces had participated in the "short and quick" assault.
The base lies near a major highway running from Damascus to Syria's southern border with Jordan and is also near the border with neighbouring Sweida province, which is largely regime-controlled.
"The base is also an important infantry base, from which the regime attacked towns and villages in the south," said Diaa al-Hariri, spokesman for Faylaq al-Awwal, one of the rebel groups in the Southern Front coalition.
The Syrian Observatory for Human Rights monitoring group confirmed that opposition groups had taken the 52nd Brigade base after clashes and intense shelling that left 14 rebel fighters and 20 government forces dead.
Rebel groups control a majority of Daraa province and its capital, Abdel Rahman said.
Syria's official news agency SANA did not report the capture of the base.
But earlier, citing a military source, it said the air force had struck the area, killing at least 40 "terrorists," who it accused neighbouring Jordan of backing.
The fall of the base is the latest in a string of defeats for the Syrian regime, which has lost territory to both rebel alliances and the jihadist Islamic State group.
It has lost the city of Palmyra and the surrounding region to the Islamic State group, which now controls around half of Syria's territory.
The loss of Brigade 52 also follows earlier defeats for the regime in Daraa, where it last month lost control of the Nasib border post, its last crossing with Jordan.
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