Salim Hamdan, Osama bin Laden's former driver, has pleaded not guilty to charges of conspiracy and providing material support for terrorism.
Hamdan, who appeared in court on Monday in a khaki prison jumpsuit, could face life in prison if convicted by the military tribunal.
The Yemeni national's trial is expected to last several weeks.
Hamdan is the first detainee to be tried before a military tribunal at the US base in Guantanamo Bay, Cuba.
The prosecution alleges Hamdan was affiliated with al-Qaeda network's inner circle and claims he was captured in Afghanistan in November 2001 - shortly after the US-led invasion of the country - with two surface-to-air missiles in his car.
However, Hamdan's lawyers say he was only a driver and mechanic in bin Laden's extended circle and needed the $200 monthly salary he was given.
In June this year the US supreme court ruled that detainees held in Guantanamo Bay could challenge their detention in civilian courts.
However, a federal judge last week rejected a request from Hamdan's lawyers to delay his trial so he could challenge his detention.
The trial is expected to take from three to four weeks, with testimony from around two dozen Pentagon witnesses.
The jury will be selected from a pool of 13 US military officers flown in from around the world and will have at least five members.
Before the trial, Keith Allred, the presiding judge, threw out some statements made by Hamdan to interrogators at the US air base in Bagram, Afghanistan and in Panjshir valley, also in Afghanistan.
Hamdan has been held at the facility since 2002, mostly in isolation.
Defence lawyers working for many of the roughly 260 detainees still being held at Guantanamo Bay say many of their clients' statements to interrogators were coerced.
Earlier this month five men, including the alleged mastermind Khalid Sheikh Mohammed, were charged with crimes relating to the September 11, 2001, attacks, which killed almost 3,000 people.
Lawyers for Hamdan plan to call Mohammed and a second of those charged, Walid bin Attash, as witnesses in his trial to support his contention that he was not a member of al-Qaeda.