Philip Markoff, 23, was indicted at Boston's Suffolk Superior Court for the April 14 murder of Julissa Brisman, a 25-year-old masseuse who advertised in the erotic services section of Craigslist.com.
The former Boston University student was ordered held without bail after his plea.
Police said Markoff used prepaid cellphones and temporary email addresses to make appointments with victims who had advertised on Craigslist, an online bazaar with listings for just about anything -- from apartments to cars to jobs.
They said he struck Brisman in the head and then shot her three times at close range at a Boston hotel.
He also pleaded not guilty to charges of armed robbery, kidnapping and weapons violations related to an April 10 assault on another woman who advertised on Craigslist. He faces additional charges for a similar attack in Rhode Island.
Detectives found the murder weapon in a hollowed-out medical textbook in Markoff's apartment, prosecutors said at his arraignment. They also discovered plastic ties similar to those used to bind the women, ammunition, disposable phones and computer evidence of emails to Brisman, prosecutors added.
The attacks prompted authorities to seek curbs on sex-related listings on Craigslist, which generates more than 20 billion page views per month in 50 countries.
Under pressure from a 40-state task force, the website dropped its erotic services section last month, replacing it with an adult services category where advertisements are individually screened by Craigslist staff.
The attacks followed the killing of George Weber, a New York reporter knifed to death after responding to a personal ad he placed on Craigslist in March, and the early-April sentencing of Michael Anderson, a Minnesota man convicted of killing a woman who responded to a babysitting ad.
Markoff's trial is scheduled for June next year.
Craiglist is partly owned by online auctioneer eBay, which bought a 25 percent stake in 2004.
(Editing by Jason Szep and Eric Walsh)