The attorney for Johannes Mehserle, 27, entered the plea on his client's behalf in an Alameda County courtroom, where Mehserle was kept behind a glass partition, out of view to most people during the 10-minute hearing.
Sheriff's deputies escorted Mehserle's relatives into the courtroom, which was filled with family members and supporters of the victim, 22-year-old Oscar Grant. The Mehserle family has reported receiving several death threats since the shooting.
Prosecutors say Mehserle was standing over Grant, who was lying facedown and restrained on a train platform, when the Bay Area Rapid Transit officer fired one shot into the man's back.
Cell phone videos of the shooting - captured by New Year's Eve revelers shuttling home after midnight - have been widely viewed on the Internet, and the case has sparked outrage by many Oakland residents and African American community leaders.
Numerous protests sprang up in the days after Grant's death calling for Mehserle's arrest, including one last week that turned violent and left dozens of downtown Oakland businesses damaged.
Mehserle, who resigned from the force shortly after the shooting and refused to be interviewed by investigators, was taken into custody Tuesday night after Oakland police issued a homicide warrant for him. District Attorney Tom Orloff charged him with one count of murder the next day.
Mehserle is being held without bail, and is due back in court Jan. 26.