The five are accused of planning and executing 2001 hijacked airliner attacks on New York, Washington and Shanksville, Pennsylvania, resulting in the deaths of 2,976 people.
They are charged with terrorism, hijacking an aircraft, conspiracy, murder in violation of the law of war and other counts, and were referred to a capital military tribunal, meaning they could be sentenced to death if convicted, the Pentagon said on Wednesday.
President Barack Obama halted the previous trial and wanted them prosecuted in civilian court. Congress opposed the move and the administration was forced to shift it back to Guantánamo.
The decision to refer the case to a military commission means the five will be arraigned before a military judge at Guantánamo Bay Naval Station in Cuba within 30 days.
The US attorney general, Eric Holder, blamed lawmakers for the policy reversal, saying their decision to block funding for prosecuting the suspects in a New York court had tied the administration's hands and forced it to move to a military trial.
The American Civil Liberties Union condemned the decision on Wednesday to proceed with a military trial.
"The Obama administration is making a terrible mistake by prosecuting the most important terrorism trials of our time in a second-tier system of justice," ACLU executive director Anthony Romero said in a statement.
"Whatever verdict comes out of the Guantánamo military commissions will be tainted by an unfair process and the politics that wrongly pulled these cases from federal courts, which have safely and successfully handled hundreds of terrorism trials," he said.
