Leaders of the demonstration drew links between the missile attacks and the supply line, saying the equipment was being used for attacks on Pakistani soil and vowing to shut down the convoys.
"We will no longer let arms and ammunition pass through ... and reach the hands of American and NATO forces in Afghanistan," Sirajul Haq, the provincial head of hardline Islamist party Jamaat-e-Islami, told the crowd. "They are using the same against our innocent brothers, sisters and children."
The supply line - along which gear passes from the Pakistani port city of Karachi and through the Khyber Pass - has increasingly come under assault, leading U.S. and NATO forces to scout possible alternative routes.
Hundreds of vehicles, including Humvees allocated for the Afghan army, have been torched in recent weeks in arson attacks on terminals, leaving several security guards dead. The convoys also are targets in Afghanistan, despite armed escorts.
Thursday's rally appeared to be the largest against Western use of the route since Pakistan's civilian government took office in March and one of the largest anti-government protests so far.
Banner-toting demonstrators chanted "Down with America" and "Jihad is the only solution of America" as they marched along a key road in Peshawar, led by party leader Qazi Hussain Ahmed.
"If America continues atrocities against Muslims, it will also not be able to live in peace," Express television showed Ahmad saying.
The protest ratchets up pressure on the new government at a time when it is also dealing with a tanking economy and the fallout over the Mumbai terror attacks that killed more than 160 people.
Pakistan's main stock market index plunged to its lowest level in more than three years Thursday, as tensions with New Delhi appeared to be rising. India ordered cricket officials to cancel next month's scheduled tour of Pakistan - a blow to the sport, which had been used to help with rapprochement between the two countries.
Pakistan also summoned an Indian envoy for an official protest over last weekend's reported incursions into Pakistani airspace by Indian aircraft after Islamabad initialized downplayed the incidents.
India says the Pakistan-based Lashkar-e-Taiba militant group was behind November's attacks in the Indian financial capital. Pakistan has arrested some suspects and clamped down on a charity, Jamaat-ud-Dawa, allegedly linked to the outlawed group, but it insists it needs evidence from India. The crackdown on the charity also has stirred anti-government sentiment, with a series of recent demonstrations on behalf of Jamaat-ud-Dawa.