The November/December 2017 issue shows a side profile of Hefner from when he was photographed by Larry Gordon in 1965, at the start of his career.
The late ladies man was 39-years-old in the photo.
Hefner will be the 11th man to ever be on the cover of the magazine - but the first to be on the cover by himself and without a semi-nude model pictured alongside.
The 91-year-old died on September 27 - two days before the initial issue was going to go to press, according to an issue from Playboy.
According to his death certificate, he died of heart failure after contracting an aggressive strain of e-coli which was resistant to drugs, it has been revealed
The certificate, which was first obtained by The Blast, lists the mogul's causes of death as cardiac arrest, respiratory failure, septicemia and e-coli.
The Los Angeles County Department of Public Health could not determine how Hefner contracted the deadly bacteria.
The first six pages of next month's issue of Playboy are also dedicated to Hefner's life.
In one article, Hef's son Cooper writes about his legendary father.
Cooper, 26, is the COO of Playboy. He stands to inherit a fourth of his father's $43million fortune.
Hefner died at the Playboy Mansion in Los Angeles on September 27 surounded by friends and family, and was laid to rest at Westwood Village Memorial Park in LA, next to the legendary sex icon Marilyn Monroe.
Monroe was on the cover of the very first edition of Playboy which Hefner published in 1953. He bought the crypt next to hers in the cemetery before he died for $75,000.
'He is in the place he was always certain he wanted to spend eternity,' Crystal Hefner told PEOPLE after he died.
The 31-year-old final wife of Hefner said he was an 'American hero,' a 'pioneer,' and a 'kind and humble soul who opened up his life and home to the world.'
Harris, who was kept out of inheriting any of the business by an iron clad prenup still managed to inherit a $7million home from her husband's passing.
She has claimed that before her husband's death that Viagra caused him to lose his hearing.
Hefner published the very first issue of Palyboy in December of 1953 after borrowing money from family members and putting the furniture in his home up for collateral for a loan from the bank.
That same year he also launched the media and lifestyle company Playboy Enterprises - which he was a boardmember of until he died.
During his life Hefner He four children from two marriages before wedding Crystal Harris, his third wife, in 2012.
Playboy is also issuing a 100-plus page tribute to Hefner's life at a time in the near future.
