The protest was the largest so far against the reopening of the routes. Shops closed early in Islamabad and police set up barricades and cordoned off roads.
Pakistan suspended NATO supply routes to Afghanistan last November after a cross-border NATO air attack killed 24 Pakistani soldiers. They were re-opened last week after U.S. Secretary of State Hillary Clinton apologised for the strike.
A survey by the Pew Research Center last month found that three out of four Pakistanis consider the U.S. an enemy, up from 69 percent last year and 64 percent three years ago.
Relations have been hurt by a series of events – the border strike, the killing of Osama bin Laden in a unilateral U.S. raid on Pakistani soil, and the fatal shooting of two armed Pakistanis by a CIA contractor.
The march was organised by the Defence of Pakistan Council, an alliance of religious political parties and organisations campaigning for a break in ties with the United States and India.
One of the group’s main leaders is Hafiz Saeed, whom India suspects of masterminding attacks on India’s financial capital Mumbai and its parliament. Saeed denies any involvement in the attacks.