Harris told Howard Stern in a recent interview that sex with the 85-year-old lasted "like, two seconds", claiming that the pair only slept together once.
Hefner took to Twitter to respond to the accusations, revealing that he has intercourse regularly.
"For the record, I have sex on a weekly basis," Hefner wrote. "And I did throughout my two-and-a-half years with Crystal."
The Playboy founder went on to reveal that he has enjoyed sex with many partners over the past decade.
"My sex life involves more than one partner and has since the end of my marriage in 1998," Hefner added.
Hefner and Harris became engaged in December 2010, but called off the engagement just days before the June wedding.
Harris has since claimed that marrying Hefner was not the "right thing" for her to do.