Playboy Enterprises Inc said he passed away peacefully at home, from natural causes.
Hefner began publishing Playboy in his kitchen at home in 1953. It became the largest-selling men's magazine in the world, shifting seven million copies a month at its peak.
Cooper Hefner, his son, said he would be "greatly missed by many".
He paid tribute to his father's "exceptional and impactful life as a media and cultural pioneer," and called him an advocate for free speech, civil rights and sexual freedom.
Hefner's trailblazing magazine helped make nudity respectable in mainstream publications, despite emerging at a time when US states could legally ban contraceptives.
It also made him a multi-millionaire, spawning a business empire that included casinos and nightclubs.
The first edition featured a set of nude photographs of Marilyn Monroe that Hefner had bought for $200. They had originally been shot for a 1949 calendar.
The silk pyjama-clad mogul became famous for his hedonism, dating and marrying Playboy models. In his later years he threw decadent parties at his luxurious mansions in Chicago and Los Angeles.
He claimed to have slept with more than 1,000 women, and credited the impotence drug Viagra with maintaining his libido.
From 2005-10, a reality TV show called "The Girls Next Door" showcased Hefner's libertine lifestyle - and the harem of young blonde women who shared it.
In 2012, aged 86, he married his third wife Crystal Harris - who was 60 years his junior.
Though critics saw Playboy as a byword for sleaze, its founder - who was born into a strict Methodist family - never shared that view.
"I've never thought of Playboy quite frankly as a sex magazine," Hefner told CNN in 2002. "I always thought of it as a lifestyle magazine in which sex was one important ingredient."
FollowPlayboy ✔@PlayboyAmerican Icon and Playboy Founder, Hugh M. Hefner passed away today. He was 91. #RIPHef8:43 AM - Sep 28, 2017 2,5042,504 Replies 118,824118,824 Retweets 129,994129,994 likes
American Icon and Playboy Founder, Hugh M. Hefner passed away today. He was 91. #RIPHef
End of Twitter post by @Playboy
Hefner faced obscenity charges in 1963 for publishing and distributing Playboy, but the jury was unable to reach a verdict.
The magazine's most significant interviewees included civil rights leader Martin Luther King Jr, Beatle John Lennon, and Cuban revolutionary Fidel Castro.
Its huge sales were certainly driven by glossy colour pictures of nude "playmates", but it also developed a reputation for fine writing, with Norman Mailer, Kingsley Amis, Kurt Vonnegut, James Baldwin, Vladimir Nabokov and Ray Bradbury among its contributors.
Their contributions allowed men to say they did not buy the magazine only for the pictures.