After several days of fighting, the insurgents took full control of Tishrin dam on the Euphrates river, a route that connects the northern provinces of Aleppo and Raqa, the Syrian Observatory for Human Rights said.
A resident of nearby Manbij confirmed the report, adding that employees of the six-turbine hydropower dam were continuing operations.
"The capture of the Tishrin dam is very important. It means that the army basically has only one road left to Aleppo," Observatory director Rami Abdel Rahman told AFP by telephone.
"The highway crossing over the Tishrin dam was the last main route from Raqa province under regime control. Now it is impassable," he said, noting that another small bridge exists further north but the road is difficult.
Another main route further south is dotted with regime and rebel checkpoints.
With the overnight capture, the rebels now hold sway over a wide expanse of territory between the two provinces bordering Turkey, which backs the revolt against President Bashar al-Assad.
The army must now rely on the Damascus-Aleppo highway to bring reinforcements to Syria's embattled commercial hub of Aleppo, where fighting is deadlocked.
Further west, a warplane launched three bombs or rockets at a rebel command centre in Atme near the Turkish border on Monday, without causing casualties or hitting its target, an AFP journalist said.
The village, a nerve centre of the rebellion two kilometres (1.2 miles) from Turkey, was once home to 7,000 inhabitants who have largely fled.
In a sign of growing confidence, rebel officers have formed a commission to lay the groundwork for a future army and liaise with the political opposition on issues such as arming fighters on the ground, a spokesman said on Monday.
He said the Free Officers Assembly will seek "to lay the correct foundations for the construction of the new Syrian army, which will be a non-partisan defender of the rights and dignity of the people," working with the opposition National Coalition.
Faced with an increasingly offensive revolt, the regime has been reducing its territorial ambitions to focus on Damascus, central Syria and Alawite bastions, as it digs in for a long war, analysts say.
As part of this effort, troops have been bombing rebel positions in the outskirts of the capital, including in Daraya, the site of the worst massacre in the 20-month conflict.
The Syrian National Council, a leading opposition group, on Monday said more than 130 people have been killed in the town in three weeks. According to the Observatory, rebels account for 80 percent of the deaths.
Pro-government daily Al-Watan said on Monday that troops were advancing on Daraya and had inflicted heavy losses on "Al-Qaeda terrorists".
Prime Minister Wael al-Halaqi, meanwhile, laid the foundation stone for a new housing project in Damascus, vowing that " despite all the challenges facing Syria, the development process will continue."
The Observatory, which relies on a network of activists and medics for its information, gave an initial toll of 19 people killed on Monday. It has recorded a total of more than 40,000 deaths in the Syrian conflict.