Sulaiman Abu Ghaith entered the plea through a lawyer to one count of conspiracy to kill Americans after being captured in Jordan over the past week.
The case marks a legal victory for President Barack Obama's administration, which has long sought to charge senior a Qaeda suspects in American federal courts instead of military tribunals at the detention center at Guantanamo Bay, Cuba. However, Republicans in Congress do not want high-threat terror suspects brought into the United States.
According to the Department of Justice, Ghaith was the spokesman for al Qaeda. He worked alongside bin Laden and current leader Ayman al-Zawahri since at least May 2001. He is also a former mosque preacher and teacher.
The day after the Sept. 11 attacks, prosecutors say he appeared with bin Laden and al-Zawahri and called on the "nation of Islam" to battle against Jews, Christians and Americans.
A "great army is gathering against you," Abu Ghaith said on Sept. 12, 2001, according to prosecutors.
Shortly afterward, Abu Ghaith warned in a speech that "the storms shall not stop - especially the airplanes storm" and advised Muslims, children and al Qaeda allies to stay out of planes and high-rise buildings. In one video, he was sitting with bin Laden in front of a rock face in Afghanistan.
Abu Ghaith was born in Kuwait, which stripped him of his citizenship after Sept. 11.
As the search for bin Laden intensified in 2002, prosecutors say Abu Ghaith was smuggled into Iran from Afghanistan.
Abu Ghaith's trial will mark one of the first prosecutions of senior al Qaeda leaders on U.S. soil. Charging foreign terror suspects in American federal courts was a top pledge by Mr. Obama shortly after he took office in 2009, aimed, in part, to close Guantanamo Bay.
Republicans have fought the White House to keep Guantanamo open. Several GOP lawmakers on Thursday said Abu Ghaith should be considered an enemy combatant and sent to Guantanamo.
Generally, Guantanamo detainees have fewer legal rights and due process than they would have in a court in America but could potentially yield more information to prevent future threats.
The Associated Press contributed to this report.