As each of the 12 counts of murder and attempted murder against him was read out in court, Alexandre Bissonnette, 28, murmured: "Not guilty."
The rampage on January 29, 2017 was one of the deadliest ever against Muslims in a Western country, leaving six dead and 19 injured.
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The gunman opened fire at the end of Sunday prayers at Quebec City's Islamic Cultural Center, a mosque located in a normally quiet neighborhood.
Police say Bissonnette called an emergency line after the shooting and confessed. They found him in his car, parked on the side of a rural road about 20 kilometers (12 miles) from the mosque.
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Judge François Huot has ordered a publication ban on evidence to be presented at the trial.
The accused appeared to show little interest in the court proceedings, only occasionally glancing in the direction of his lawyers.
Over the coming days, the court will select 12 jurors for the trial by judge and jury, from a list of 600, of which nearly one-fifth have asked to be recused.
The prosecution and defense are expected to then present a joint statement of facts, followed by the first witnesses expected to testify.
The trial is scheduled to last until the end of May.
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