Robert Bowers, suspected of killing 11 people in the Tree of Life synagogue in Pittsburgh, Pennsylvania, has pleaded not guilty.
The truck driver entered the plea during an appearance in federal court for charges that could result in the death penalty.
There are 44 charges against the avowed antisemitic ranging from murder to hate crimes and obstructing the practice of a religion.
Prosecutors have said they will be pursuing the death penalty against him in the incident that is the deadliest-ever attack against Jews in the US.
He appeared in court with a bandage on his left arm, the result of a shootout with police at the synagogue on the morning of 28 October.
Mr Bowers is accused of interrupting a bris at the synagogue, located in the Squirrel Hill neighbourhood of Pittsburgh, and gunning down victims using a semiautomatic rifle and handguns after shouting "All Jews must die."
Donning a red prison-issued jumpsuit, he did not speak much in court except to say he understood the charges against him could result in being put on death row and to enter his plea.
During his previous court appearance earlier in the week, Mr Bowers had been shackled to a wheelchair since he was still being treated for his gunshot wound.
The 11 victims were regulars at the Tree of Life, there to attend Saturday services when the massacre occurred.
Rose Mallinger was the oldest among them at 97 and "was considered a pillar of the Jewish community" according to a statement from her family, Jerri Rabinowitz was a doctor who was killed as he ran towards the gunfire to help the wounded, Cecil and David Rosenthal were brothers and both developmentally disabled. One congregant Katy Levin told ABC News the pair were always seen together and she took comfort they were next to each other in their last moments.
Daniel Stein was a new grandfather, Richard Gottfried was a dentist and once-president at the Tree of Life, Joyce Fienberg was a grandmother of six who had mastered social media in order to stay in touch with them, 88-year-old Melvin Wax would lead services for the congregation when they needed it.
Bernice and Sylvan Simon, both in their late 80s, were killed in the same synagogue in which they were married and Irving Younger was another grandfather and volunteer who greeted his congregation at services.
Judah Samet, 80, was not among the victims having arrived at the synagogue just four minutes late. As a child, Mr Samet had escaped the Bergen-Belsen concentration camp in Germany during the Second World War.
“I survived the second time," he told the Washington Post about how he was in the parking lot just as the shooting was taking place.
Four police officers were also wounded in the shooting.
Critics have pointed out Mr Bower's antisemitic views openly expressed on social media channels like Gab were driven in part by the rhetoric of Donald Trump.
