Patrick Crusius, 21, is accused of carrying out the August 3 mass shooting at a Walmart in the west Texas city of El Paso. He confessed to police upon his arrest and said he was targeting "Mexicans."
"Crusius pleaded not guilty this afternoon," the El Paso district attorney's office said.
"District Attorney Jaime Esparza will seek the death penalty."
Texas man to be arraigned in El Paso mass murder, accused of targeting Mexicans
The hearing in the El Paso County Courthouse was Crusius's first public appearance since his arrest.
Texas prosecutors charged Crusius with capital murder in August, the day after the attack.
The United States (US) attorney for the Western District of Texas, John Bash, then added that the case was being treated as an act of domestic terrorism.
Crusius is believed to have driven nine hours from Allen, his hometown in North Texas, to the majority-Hispanic border city of El Paso.
He can be seen on video from the Walmart security cameras entering the store with an assault rifle.
Mexico does not want El Paso shooter executed
Prior to carrying out the attack, Crusius published a white supremacist manifesto online which expressed fears of a "Hispanic invasion" of Texas.
Eight of the people killed in the rampage were Mexican nationals, and many of the other victims were Americans of Hispanic origin.
Donald Trump's critics have accused the president of stoking white nationalist hatred in the US with anti-immigrant rhetoric, including comments referring to Mexican immigrants as criminals and rapists.
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