James entered the plea Friday in federal court in Brooklyn.
He's charged with committing a terrorist attack or other violence against a mass transportation system and discharging a firearm during a violent crime. Both counts carry a maximum sentence of life in prison.
U.S, District Judge William F. Kuntz began the proceeding by asking James, "How are you doing today?"
"Pretty good," James responded.
Authorities say that James drove from Philadelphia and unleashed smoke bombs and dozens of bullets in a train full of morning commuters as it approached a Brooklyn station. The shooting victims ranged in age from 16 to 60; all survived.
Authorities said James’s bank card, phone, and a key to a van he had rented were found at the shooting scene. Police also said they found the 9mm Glock semiautomatic handgun used in the shooting and traced it to James.
Defense attorney Mia Eisner-Grynberg had cautioned at the time of James' arrest not to rush to judgment and noted that James alerted police to his whereabouts. He was arrested in Manhattan’s East Village after he called a tip line saying he was at a fast food restaurant in that section of the city.
Eisner-Grynberg declined comment outside court Friday.
James was arrested about 30 hours after the attack.
His attorneys previously said his rights were violated by the FBI after his arrest. They argued that FBI agents took oral DNA samples from Frank James without informing his legal team.
They also say he was improperly questioned in his jail cell and claim agents had him sign several documents.
James, 62, is accused of setting off smoke grenades and firing a handgun 33 times inside a crowded subway train on April 12.
James was allegedly wearing a surgical mask, a construction helmet, and a safety vest when he opened fire inside a Manhattan-bound N train in Sunset Park.
The Associated Press contributed to this report.