Hefner, 86, Tweeted the news of her passing to his Playboy family late Sunday night.
"Mary passed away today. We loved her more than words could say," he wrote of the woman who, by some accounts, marked one of his longest female relationships.
O'Connor, who had been by Hefner's side through more than four decades and three marriages and had counseled dozens of aspiring bunnies who lived at his California mansion, became more well known in recent years as she was seen on the shows The Girls Next Door and Kendra. There she gave astute advice to a host of scantily clad girls, who sought her wisdom on love, life and all things Hef.
The news of her passing drew kind words of praise from reality show stars and Playmates Holly Madison, Kendra Wilkinson and Bridget Marquardt, as well as Jenny McCarthy and Hefner's newest wife Crystal Harris.
"R.I.P. Mary O Connor," McCarthy writes. "She was Hef's secretary for 50 years. She was our 'den mom' and angel in the house. She will be missed."
"Rest in Peace Mary," wrote Madison on Twitter.
"She was an amazing person who helped me through so much," Wilkinson Tweeted. "But I know she's happy with her boo Captain Bob."
Added Harris: "It's a very sad day here at the Playboy Mansion. We love you Mary."