US Defence Secretary Lloyd Austin has revoked a pre-trial agreement reached with men accused of plotting the 11 September 2001 attacks.
In a memo on Friday, Mr Austin also said he was revoking the authority of the officer overseeing the military court who signed the agreement on Wednesday.
The original deal, which would reportedly have spared the alleged attackers the death penalty, was criticised by some families of victims.
The 9/11 attacks in New York, Virginia and Pennsylvania were the deadliest assault on US soil since the 1941 Japanese attack on Pearl Harbor, Hawaii, where 2,400 people were killed. They sparked the "War on Terror" and the invasions of Afghanistan and Iraq.
In his memo, Mr Austin named five defendants including the alleged ringleader of the plot, Khalid Sheikh Mohammed, all of whom are held in Guantánamo Bay, Cuba. The original deal named three men.
"I have determined that, in light of the significance of the decision to enter into pre-trial agreements with the accused... responsibility for such a decision should rest with me as the superior authority," Mr Austin wrote to Brig Gen Susan Escallier.
"I hereby withdraw your authority. Effective immediately, in the exercise of my authority, I hereby withdraw from the three pre-trial agreements."
The White House said on Wednesday that it had played no role in the plea deal.
The five men named in the memo were: Khalid Sheikh Mohammed, often referred to as KSM, Walid Muhammad Salih Mubarak bin Attash, Mustafa Ahmed Adam al-Hawsawi; and two others not mentioned in the original plea: Ramzi bin al-Shibh and Ali Abdul Aziz Ali.
The men have been in custody for decades without trial. All have alleged they were tortured - KSM was subjected to simulated drowning, so-called "waterboarding", 183 times before it was banned by the US government.
All have already faced more than a decade of pre-trial hearings, complicated by the allegations and evidence of torture against them.
Several family members of victims had criticised the terms of the deal struck on Wednesday as too lenient.
Brett Eagleson, the president of 9/11 Justice, which represents survivors and relatives of victims, had told the BBC earlier this week that the families were "deeply troubled by these plea deals".
Speaking on Saturday, Terry Strada, who lost her husband Tom and chairs the 9/11 Families United group, told the BBC she was "very pleased" to see the Pentagon revoke the plea deal and put the death penalty back on the table.
If the men are found guilty after a trial, Mrs Strada said she would want to see the death penalty, "not because I am ghoulish or a horrible person, it's because it fits the crime".
"They've murdered nearly 3,000 Americans on American soil... lives were just permanently altered on that day," she added.
A lawyer at Guantanamo representing Mohammed told The New York Times that he was shocked by the sudden u-turn.
“If the secretary of defence issued such an order, I am respectfully and profoundly disappointed that after all of these years the government still has not learned the lessons of this case,” said lawyer Gary Sowards.
“And the mischief that results from disregarding due process and fair play.”
The men have been accused of a litany of charges, including attacking civilians, murder in violation of the laws of war, hijacking and terrorism.
In September, the Biden administration reportedly rejected the terms of a plea deal with five men held at the US Navy base in Cuba, including Mohammed.
The men had reportedly sought a guarantee from the president that they would not be kept in solitary confinement and would have access to trauma treatment.
KSM is alleged to have brought the idea of hijacking and flying planes into buildings to al-Qaeda leader Osama bin Laden. He was captured in Pakistan in 2003 along with Hawsawi, a Saudi who was an alleged fundraiser.
Ali, a computer scientist and nephew of KSM, is accused of providing technical support to the 9/11 operation.
Bin al-Shibh, a Yemeni, allegedly co-ordinated the attacks and had planned to be a hijacker but could not secure a US visa.
Bin Attash, also a Yemeni, is accused of bombing the USS Cole in Yemen in 2000, which killed 17 sailors, and involvement in the 11 September attacks.
Several Republicans applauded the defence secretary for revoking the deal.
Speaker of the House Mike Johnson said the "Biden-Harris Administration is correct to reverse course", which he said followed Republicans "launching investigations into this terrible plea deal".
"Now deliver long awaited justice for 9/11 families," he said.
South Carolina Senator Lindsey Graham said the decision "exercised good command judgement".
"The previous plea deal would have sent absolutely the wrong signal to terrorists throughout the world," he added.
Earlier on Friday, Republican Chairman of the House Armed Services Committee Mike Rogers demanded answers from Mr Austin on how the deal was struck.
“This deal signals willingness to negotiate with terrorists who deliberately harm Americans,” he wrote in a letter to the defence secretary.
