Mr Ghailani's trial will be an important test case for closing Guantanamo
The first Guantanamo detainee to be brought to the US for trial has pleaded not guilty to involvement in two embassy blasts in East Africa in 1998.
Ahmed Ghailani appeared before a federal court in New York, after being transferred there earlier in the day.
Mr Ghailani, a Tanzanian, was detained in Pakistan in 2004 and taken to Guantanamo in late 2006.
The case is seen as a test of the Obama administration's pledge to close Guantanamo Bay by next January.
Mr Ghailani entered the courtroom in Manhattan wearing a blue prison uniform.
AHMED KHALFAN GHAILANI
Born in Zanzibar but date varies from 1970 to 1974
Alleged to have been Osama Bin Laden's bodyguard
Accused of buying equipment for embassy attack in Tanzania in 1998 and involvement in simultaneous Kenya attack
Indicted in 1998 in New York and reportedly fled to Afghanistan
Reported in Liberia in 2001
Arrested in Pakistan in 2004
Judge Loretta Preska asked him for his plea to charges of conspiring to commit the bombings of US embassies in Tanzania and Kenya that killed 224 people, including 12 Americans.
"Not guilty," Mr Ghailani said.
Judge Preska set a date of 16 June for the next hearing.
The justice department says Mr Ghailani faces 286 charges.
They include conspiring with Osama Bin Laden and other members of al-Qaeda to kill Americans around the world, and murder charges for each of the victims of the embassy attacks of 7 August 1998.
If found guilty Mr Ghailani could face the death penalty.
Earlier, US Attorney General Eric Holder said in a statement: "With his appearance in federal court today, Ahmed Ghailani is being held accountable for his alleged role in the bombing of US embassies in Tanzania and Kenya."
The BBC's Kevin Connolly in Washington says if President Barack Obama is to honour his promise to close Guantanamo Bay in his first year in office he needs as many cases from there as possible to be tried as ordinary criminal cases in federal courts.
However, the US Congress has already rejected an administration request for funding to close down Guantanamo amid widespread opposition to bringing detainees on to the US mainland.
Our correspondent says Mr Ghailani's case will also establish whether defence lawyers will seek to have US federal trials thrown out on the grounds that the government has admitted applying harsh interrogation techniques to some detainees, and holding others in secret prisons overseas.
Mr Obama is hoping to persuade America's allies around the world to take some of the other Guantanamo detainees, but negotiations have proved difficult.
A number of high-value prisoners are likely to face indefinite detention without trial, our correspondent says.
According to the transcript of a closed-door hearing in March 2007, Mr Ghailani admitted delivering explosives used to blow up the US embassy in the Tanzanian city of Dar es Salaam.
Ahmed Ghailani appeared at the Federal Courthouse in New York
However, he told the hearing he did not know about the attack beforehand and apologised to the US government and the victims' families.
Investigators say he left Africa just before the bombings.
Mr Ghailani is thought to have been born on the Tanzanian island of Zanzibar in 1970 or 1974 - making him 39 or 35 years old. He is said to speak fluent English.
He is alleged to have risen through the ranks of al-Qaeda to become a bodyguard of Osama Bin Laden.
According to the US transcript, he admitted visiting an al-Qaeda training camp in Afghanistan after the bombings. But he denied being a member of al-Qaeda.
Analysts described him as a very important figure, who was probably sent to east Africa at the time of the bombings by Osama Bin Laden's second-in-command, Ayman al-Zawahiri.
It is suggested that Mr Ghailani fled to Afghanistan after being indicted in 1998.