Dr Conrad Murray, a Houston cardiologist who was with Jackson when he died in June last year, appeared in court in Los Angeles on Monday.
Jackson hired Murray to be his personal physician as he prepared for a strenuous series of comeback performances.
Officials say the singer, 50, died after Murray administered the powerful general anaesthetic propofol and two other sedatives to get the chronic insomniac to sleep.
Jackson's father Joe, mother Katherine, and siblings LaToya, Jermaine, Tito, Jackie and Randy were in court to hear an attorney for Murray enter the not guilty plea.
The Los Angeles County District Attorney's Office charge that Murray "did unlawfully, and without malice, kill Michael Joseph Jackson ... in the commission of an unlawful act."
Murray, who could face up to four years in prison if convicted, has acknowledged giving the anesthetic propofol to Jackson following his "repeated demands/requests" for the drug.
Powerful anaesthetic
Propofol is a potent anesthetic used to render patients unconscious before major surgery. Medical experts say it should only be used and administered by trained staff under hospital conditions.
The Los Angeles County's coroner's office blamed Jackson's death on "acute propofol intoxication".
Monday's long-anticipated hearing took place in a circus-like atmosphere, as Jackson devotees gathered outside the court to protest the manslaughter charge, saying Murray should have been charged with murder.
"We want justice for Michael," said Michelle Perell, 26. "He should be charged with first or second degree murder and go to jail for life. He killed the most beautiful person in the world."
Other fans brandished banners which read: "The World Wants Justice for Michael" and: "Conrad Murray is a Murderer: Arrest him in handcuffs."
Brian Oxman, a lawyer for Jackson's family, has also criticised the involuntary manslaughter charge, saying it amounted to a "slap on the wrist".
'Slap on the wrist' claim
"I don't think it would satisfy anybody, the millions of fans around the world," he told CBS television.
"That is just a slap on the wrist and a slap in the face because Michael Jackson was someone who we knew was in danger of being brought to his knees, brought to his death by the use of these medications."
The manslaughter case against Murray was built during a prolonged investigation which saw raids on the doctor's offices in Houston and Las Vegas.
According to affidavits unsealed during the investigation, Murray told investigators he feared Jackson was becoming addicted to propofol and that he was trying to wean him off the drug at the time of his death.
Murray allegedly told investigators he left the singer alone for a few minutes before returning to find he was not breathing.
Murray has insisted he didn't give Jackson anything that should have caused the singer's death.
In a one-minute video posted on YouTube in August, Murray assured his patients that he had told the truth and said he had "faith the truth will prevail".
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