The goalkicking wing impressed as the Wallabies' number 10 against the Barbarians at Twickenham last weekend, and coach Robbie Deans has retained him in that pivotal role.
O'Connor, though, has big boots to fill as he takes over from Australia's World Cup fly-half Quade Cooper, who is currently injured.
Elsewhere, centre Berrick Barnes has recovered from a blow to his back to start alongside Rob Horne in midfield, while lock Rob Simmons replaces Nathan Sharpe.
Simmons for Sharpe is the only change up-front from when the countries last met 41 days ago in the World Cup bronze medal match at Eden Park, when Australia triumphed 21-18.
Wales will be keen to avenge that defeat, which is a fact not lost on Deans.
"(Wales coach) Warren (Gatland) will be looking for his group to push on from the World Cup," Deans said.
"And this match provides him with a perfect opportunity to do just that as a springboard heading into the next Six Nations."
Australia team to play Wales in Cardiff on Saturday:
A Ashley-Cooper; L Turner, R Horne, B Barnes, D Ioane; J O'Connor, W Genia; J Slipper, T Polota-Nau, S Ma'afu, J Horwill (capt), R Simmons, S Higginbotham, D Pocock, B McCalman.
Replacements: S Moore, B Alexander, N Sharpe, R Samo, B Lucas, B Tapuai, A Fainga'a.
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