Castro didn't say anything at his arraignment Wednesday morning in Cleveland. His attorneys announced the not-guilty plea.
The 52-year-old was indicted last week on nearly 330 charges, including aggravated murder, kidnapping and rape. He is being held on an $8 million bond.
The 142-page grand jury indictment covers only the period from August 2002, when the first victim disappeared, to February 2007. Prosecutors say the investigation will continue and they are leaving the door open to pursuing a death penalty case against Castro.
The women, Amanda Berry, Gina DeJesus and Michelle Knight, disappeared separately between 2002 and 2004, when they were 14, 16 and 20 years old. Each said they had accepted a ride from Castro, who remained friends with DeJesus' family and even attended vigils over the years marking her disappearance.
The women haven't spoken publicly since their rescue last month.
Castro was arrested May 6 after Berry broke through a locked door, yelled to neighbors for help and escaped with DeJesus and Knight.
Berry, 27, told officers that she was forced to give birth in a plastic pool in the house so it would be easier to clean up. Berry said she, her baby and the two other women had never been to a doctor during their captivity.
Knight, 32, said her five pregnancies ended after Castro starved her for at least two weeks and "repeatedly punched her in the stomach until she miscarried," authorities said.
She also said Castro forced her to deliver Berry's baby under threat of death if the baby died. She said that when the newborn stopped breathing, she revived her through mouth-to-mouth resuscitation.
Castro's two brothers were arrested with him but later were cleared of involvement in the case and were released.
The Associated Press contributed to this report.