The Supreme Court ruling also leaves thousands of other officials, including Cabinet ministers loyal to President Asif Ali Zardari, facing reopened corruption and other criminal cases. That is sure to further weaken the U.S.-backed leader, who is unpopular and under pressure to give up much of his power.
"All the benefits given under the (amnesty) - cases withdrawn, acquittals made - are declared void," Chief Justice Iftikhar Mohammed Chaudhry said in announcing the decision of the 17-member bench.
Soon after, Pakistan's opposition party called for Zardari's resignation.
Presidential spokesman Farhatullah Babar told reporters that the government respected the court ruling but would issue a formal response after reviewing the judgment. He noted that, as president, Zardari has immunity from prosecution and also indicated that he would not step down.
Fresh political turmoil in Pakistan could mean uncertainty for the U.S., which relies significantly on Islamabad for support in its war on terror in the Afghanistan-Pakistan region, a Western diplomat in Islamabad, speaking on condition of anonymity, told CBS News' Farhan Bokhari.
In the wake of President Barack Obama's recently announced new strategy on Afghanistan, the U.S. wants Pakistan to step up the hunt for Taliban militants on its own side of the border.
"Growing political turmoil in Pakistan will only weaken the resolve of the government to support the U.S. If president Zardari is surviving for his political survival, can he realistically be expected to step up support for the U.S.?" an official told Bokhari.
But others said U.S. security interests are principally guaranteed by the Pakistan military, which has increasingly fought Taliban militants this year.
"I don't believe unexpected political changes in Pakistan seriously jeopardises what Pakistan is doing in promoting U.S. interests," a NATO defense official told Bokhari. "For the moment, it is hard to know for certain exactly what happens in future. But theoretically, if President Zardari is even forced to resign, his successor will want to continue working to support the U.S."
The amnesty was part of a U.S-brokered deal with former military ruler Pervez Musharraf that paved the way for former Prime Minister Benazir Bhutto to return home from self-exile and take part in politics without facing cases her party says were politically motivated. Zardari, Bhutto's husband, took control of the party after Bhutto was assassinated in 2007.
The amnesty, known as the National Reconciliation Ordinance, had angered some civil rights activists and ordinary Pakistanis who said it protected the wealthy elite who govern the impoverished, corruption-plagued nation from being punished for their alleged crimes.
Despite his immunity from prosecution, Zardari's opponents challenging his eligibility for the post, arguing that if it were not for the amnesty he would not have been able to run for president. Analysts and legal experts are divided over whether this push will succeed, and the process is likely to take months.
Zardari was democratically elected and heads the largest party in parliament. Even some of his critics argue that stopping him midterm, something that would likely require a nod from the still-powerful army, would represent a setback to Pakistan's transition to democratic rule after years of military government.
Zardari, 54, has long been haunted by corruption allegations dating back to governments led by his late wife, Bhutto. He spent several years in prison under previous administrations. He denies any wrongdoing.
The amnesty had been protecting Zardari from six graft cases dating back to the late 1990s. One case alleges he misappropriated $1.5 billion. The president's office has declared the cases "unproven politically motivated allegations."
Earlier this year, Zardari gave in to street protests and reinstated Chaudhry as the chief justice after he was fired by Musharraf. Many analysts took Zardari's reluctance to restore Chaudhry as a sign he feared the judge would try to undermine him.
Ever since Zardari took over the presidency in September 2008, the opposition has demanded he give up sweeping powers he inherited from Musharraf. Pakistan's original constitution envisioned a parliamentary system in which the presidency is a ceremonial role, but the balance of power shifted under Musharraf, who took power in a 1999 military coup.
A few weeks ago, amid mounting pressure, Zardari relinquished command of the country's nuclear arsenal and said he would give up more powers soon. But that's a promise he's made before, including in a major speech to lawmakers just days after being sworn in.
The pressures on Zardari come as the U.S. needs Pakistan's aid in the fight against the Taliban more than ever now that an additional 30,000 American troops are heading next door to Afghanistan.
The U.S. fears that unless Pakistan does more to crack down on militants on its side of the border, they will continue to find safe havens from where to plan attacks on American and NATO forces in Afghanistan.