O'Neill stepped down "for personal reasons" according to a statement released Wednesday by WinSport.
Stephen Norris, vice-president of sport, will serve as interim president and CEO, the organization said.
WinSport is formerly the Calgary Olympic Development Association.
The organization operates Canada Olympic Park, pays a large portion of the Olympic Oval's operating budget and is responsible the endowment funds invested from the profits of the 1988 Winter Games.
O'Neill took the job in December, 2010, after five years as CEO of Molson Canada.
The first and second phases of a $204-million Winter Sport Institute were completed at Canada Olympic Park during his tenure. The third and final phase is scheduled to open in April.
"I have been an advocate, supporter and significant funder of amateur and professional sport all of my life and the opportunity to make an impact with WinSport has been personally rewarding," O'Neill said in the statement.
"I have every confidence that Stephen will ensure our shared vision of becoming a one-stop shop for elite athletes is realized."