Saeed, the founder of the banned Lashkar-e-Taiba, told a rally held at a ground a short distance from parliament that the Pakistani people will never allow the resumption of NATO supplies and there would be protests across the country If the supply routes are reopened, "American agents will again sneak into Pakistan and start killing innocent citizens", he claimed.
"Pakistani leaders, including army chief Gen Ashfaq Parvez Kayani, should step down if they think they cannot safeguard national interests," he said as protesters shouted slogans in support of jihad.
Earlier in the day, thousands of supporters of the Defa-e-Pakistan Council marched to the ground from Aabpara and other parts of Islamabad.
Many of them carried the black-and-white flags of the Jamaat-ud-Dawah, a front for the LeT.
The protesters shouted "Down with the America-India- Israel nexus," "Death to America" and "American subjugation is unacceptable".
Some of them carried banners and placards that denounced the government's ties with the US and featured slogans like "Terrorists, the routes are closed for you".
Others who addressed the rally included Defa-e-Pakistan Council chairman Maulana Sami-ul-Haq, described as the "father of the Taliban", former Inter-Services Intelligence chief Hamid Gul and Pakistan Muslim League-Zia leader Ejaz-ul-Haq.
"We want to take Pakistan out of the so-called war on terror, which is directed against Muslims," said Sami-ul-Haq.
He warned the government of "serious consequences" if it reopens the NATO supply routes to appease the US.
The protest was organised by the Defa-e-Pakistan Council and the Jamaat-e-Islami to pressure the government not to reopen the supply routes which were closed after a cross-border NATO air strike killed 24 Pakistani soldiers in November. A joint session of parliament is set to debate recommendations for revamping Pakistan-US relations.
The parliamentary review is expected to pave the way for Pakistan to reopen the NATO supply routes after imposing a tax on container trucks and oil tankers.
Both Hafiz Saeed and Ahmed Ludhianvi, the head of the Ahl-e-Sunnat Wal Jamaat (ASWJ), participated in the rally despite an order reportedly issued by the city administration to bar them from entering Islamabad.
The ASWJ is considered a front for the banned Sipah-e-Sahaba, which has been linked to several recent sectarian attacks.
While Saeed was allowed to address the gathering, police sought to prevent Ludhianvi from speaking.
He then left the venue. Though police tried to stop him on a busy road linking Islamabad and Rawalpindi, Ludhianvi kept moving with his four-vehicle motorcade.
Pakistan had put Saeed under house arrest after the 2008 Mumbai attacks, which he was accused of masterminding.
He was freed after less than six months on the orders of the Lahore High Court.
Saeed cobbled together the Defa-e-Pakistan Council, which includes over 40 hardline and extremist groups.
The coalition has organised large rallies and protests across Pakistan that were mainly directed against the US and India.