The advance on the town of Khan Sheikhun in Idlib province comes after months of airstrikes on the area by the Syrian regime and its Russian ally.
"Regime forces took full control of the town of Khan Sheikhun and are currently clearing it of mines," said Syrian Observatory for Human Rights chief Rami Abdel Rahman.
Pro-government fighters have now "surrounded an area stretching from the south of Khan Sheikhun into northern Hama province, cutting off all roads out" for Turkish troops in the nearby town of Morek, he said.
The Observatory said 21 anti-government fighters including 18 militants were killed in Wednesday's clashes, along with 10 government or loyalist fighters.
Syria regime forces enter key town amid fierce clashes: monitor
The takeover came after days of heavy fighting against rebels and militants who control the Idlib region, which sits on the Turkish border and is the last major stronghold of opposition to the Russia-backed government of President Bashar al-Assad.
It has been administered since January by the Hayat Tahrir al-Sham alliance, which is led by militants from Syria's former al Qaeda affiliate.
The region of some three million people was supposed to be protected by a buffer zone deal signed last September by Moscow and rebel backer Ankara, but the government and Russian forces have subjected it to heavy bombardment since late April.
Around 890 civilians have been killed since then, according to the Britain-based Observatory.
More than 400,000 people have been displaced by the fighting over the same period, the United Nations says.
In recent weeks, regime forces have inched forward at the southern edges of the bastion.
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On Tuesday, anti-Assad fighters pulled back from Khan Sheikhun and the countryside to its south, said the Observatory, which has a network of sources across Syria.
Wednesday's advance raises the stakes in a showdown between Syria and Turkey, whose Foreign Minister Mevlut Cavusoglu has vowed to "do whatever is necessary to ensure the security of our soldiers and observation posts".
Moscow claimed rebel attacks against a key Russian airbase to the west of Idlib and on regime-held civilian areas had continued despite the presence of the Turkish posts.
The Syrian regime has accused Turkey of backing "terrorists", its term for both militants and rebels.
Retaking Khan Sheikhun has long been a key government objective, as the town lies on the highway connecting Damascus to second city Aleppo.
A Turkish military convoy crossed the border into Idlib on Monday and headed south along the highway, drawing condemnation from Damascus.
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Ankara alleged an airstrike had targeted its troops, while a Syrian pro-government newspaper said regime aircraft had targeted a rebel vehicle leading them.
The convoy was at a standstill on Wednesday just north of Khan Sheikhun, after government forces to the south cut the road into the town.
An AFP correspondent on Wednesday saw dozens of lorries transporting people fleeing the fighting towards northern Idlib.
"We continue to receive disturbing reports of significant displacements occurring in north-west Syria," said David Swanson, the UN's regional spokesman for the Syria crisis.
Swanson added that on Tuesday some 1,500 people who had been living in the northern Hama area were reportedly displaced northwards into Idlib.
The war in Syria has killed more than 370,000 people since it started with the brutal repression of anti-government protests in 2011.
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