Confessed mastermind Khalid Sheikh Mohammed and the four other accused opted to plead neither innocent nor guilty, but rather to defer their plea to a later date.
The special military tribunal charged Mohammed, 47, and the four others with "conspiracy, attacking civilians, murder and violation of the law of war, destruction, hijacking, terrorism" for their role in the strikes in which Al-Qaeda militants flew hijacked airliners into the World Trade Center in New York, the Pentagon, and a field in Pennsylvania.
If found guilty, the five face the death penalty for their role in the attacks that killed 2,976 people.
Mohammed was charged along with his Pakistani nephew Ali Abd al-Aziz Ali, also known as Ammar al-Baluchi; Mustapha al-Hawsawi of Saudi Arabia; and Yemenis Ramzi Binalshibh and Walid bin Attash.
After a hearing lasting more than nine hours, Mohammed and the co-defendants -- in a much-anticipated first public appearance in three years -- opted to defer their plea.
"Maybe you're not going to see us any more," Binalshibh shouted out in a dramatic moment at the arraignment hearing in the US base in southeastern Cuba, telling Judge James Pohl, "You are going to kill us."
Dressed in white jumpsuits, with some wearing white turbans, the men mostly watched the proceedings in silence, refusing to engage with the officials.
Binalshibh interrupted however by suddenly standing to pray, and then alternately kneeling and standing.
He also shouted out: "The era of Kadhafi is over but you have Kadhafi in the camp ... you are going to kill us and say that we are committing suicide."
Only one, bin Attash, was handcuffed when the group was brought into court, but Pohl ordered the manacles removed after being assured he would "behave appropriately."
The arraignment, one of the last steps before a so-called "trial of the century" takes place, marks the second time the United States has tried to prosecute the 9/11 suspects.
During the procedures the five men mostly kept their eyes fixed on the ground. Two of them were reading a book which appeared to be the Koran, while they were also passing a copy of The Economist magazine among themselves.
"Accused refused to answer," Pohl repeated over and over again, each time an accused defiantly refused to respond to questions.
Mohammed, dubbed KSM for his initials, remained calm, his long, flowing beard appearing to have been dyed with red henna.
Mohammed's lawyer David Nevin said his client, who three years ago confessed to the 9/11 attacks "from A to Z," probably would not speak at the hearing because he is "deeply concerned by the fairness of the process."
The accused men also refused to wear headphones to hear the simultaneous translation of the proceedings, which were being held in English. Their lawyers said it reminded them of their harsh interrogations.
Bin Attash's civilian attorney Cheryl Borman, the only woman on the defense team, was dressed in black and wore a hijab. "Because of what happened to them ... during the last eight years, these men have been mistreated," she argued.
The hearing comes about one year after President Barack Obama ordered the US Navy SEALs raid that killed Al-Qaeda chief Osama bin Laden.
The five men have been held for years at the US naval base in southeastern Cuba while a legal and political battle has played out over how and where to prosecute them. Debates have also raged over their treatment.
Mohammed was arrested in 2003 and spent three years in secret CIA jails where he was subjected to harsh interrogations, including waterboarding, and confessed to a series of attacks and plots.
The Pentagon opened four military bases in the United States to allow families of the 9/11 victims to watch the case unfold on a giant screen.
The trial could still be years away, unless Mohammed pleads guilty to be put to death sooner and become a "martyr" for al Qaeda.