The 35-year-old Melbourne Rebels captain will play his last game on Australian soil when he leads the Rebels out against Super Rugby champions Queensland Reds at AAMI Park on June 29.
He will then end his career in South Africa where the Rebels play their last two matches and complete their season as they are not in finals contention.
Centre Mortlock has battled calf muscle injuries this season - his second of a three-year deal with the fledgling Rebels - and only made his season start in round 12.
Mortlock played 80 Tests for the Wallabies, amassing 489 points.
At his peak, Mortlock, a powerful runner and defender, was one of the best outside centres in world rugby.
He has played more than 140 Super Rugby games, spending most of his career at the Brumbies, where he made his debut in 1998.
He remains the highest-scoring Australian in Super Rugby history, the only Australian to rack up more than 1000 points.
"There's no secret I still love what I do and still love playing. It's more about my body," said Mortlock.
"Being on the sidelines for nine of 10 weeks this season was extremely frustrating and tough. My body is back in the shape I want it to be but I think it is the right time (to retire)."
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