Ahmed Abu Khatallah pleaded not guilty, news media reported.
He was captured by US Special Operations forces inside Libya earlier this month and was transferred to the US.
Libya, however, denounced the arrest arguing that the US violated its sovereignty. Libya’s Justice Minister Saleh al-Marghani even called on Washington to return the suspect to Libya.
At the US court, Abu Khatallah spoke quietly in Arabic with a translator as he was charged with one count of providing material support to “terrorists.” A federal grand jury returned a single-count indictment Thursday.
"Now that Ahmed Abu Khatallah has arrived in the United States, he will face the full weight of our justice system," US Attorney General Eric Holder said. "We will prove, beyond a reasonable doubt, the defendant's alleged role in the attack that killed four brave Americans in Benghazi."
Abu Khatallah is charged with "conspiracy to provide material support and resources to terrorists, knowing and intending that these would be used in preparation for and in carrying out a killing in the course of an attack on a federal facility, and the offense resulted in death," according to Holder's statement.
The statement said the Justice Department can bring additional charges as the investigation still continues.
In his court appearance, Abu Khatallah requested consular assistance from the Libyan government.
In the September 2012 attack, four Americans, including Ambassador Christopher Stevens, were killed. The incident raised a political dispute in Washington, with Republicans accusing the White House of failing to respond decisively and trying to hide some facts afterward.