Tatafu Polota-Nau will not line up against the Springboks after succumbing to a hamstring niggle.
The Wallabies hooker had been "managed" through training in Port Elizabeth this week and was initially named in the side to face the Boks but failed a fitness test on Friday.
Brumbies rake Folau Faingaa will make his first Test start in his place after three appearances off the bench.
Faingaa's promotion opens up a spot for Brandon Paenga-Amosa on the bench.
The Reds hooker made his Test debut in June but this will be his first involvement in the Rugby Championship, after plying his trade in the NRC in recent weeks.
Polota-Nau returned to the Wallabies setup in the Rugby Championship after his first season with Premiership side Leicester and has every Test since coming back into the fold.
His withdrawal is the latest in a string of last-minute changes for the Wallabies through this tournament.
Taniela Tupou pulled out of the opening Bledisloe in Sydney, David Pocock, Israel Folau and Adam Coleman were late scratchings in Brisbane and captain Michael Hooper withdrew from the Gold Coast Test against Argentina a fortnight ago.
Australia is looking to break a seven-year drought in South Africa when they take on the Springboks in Port Elizabeth.
The Wallabies and Springboks drew in Bloemfontein last year but Australia has not beaten South Africa on the road since a 14-9 2011 win in Durban.
The Wallabies take on the Springboks on Saturday September 29 in Port Elizabeth, kicking off Sunday September 30 at 1:05am AEST, LIVE on FOX SPORTS and Channel Ten.
Wallabies team to face the Springboks
IN: Michael Hooper, Rob Simmons, Brandon Paenga-Amosa
OUT: Pete Samu, Lukhan Tui, Tatafu-Polota-Nau
1. Scott Sio (49 Tests)
2. Folau Faingaa (3 Tests)
3. Taniela Tupou (6 Tests)
4. Izack Rodda (11 Tests)
5. Adam Coleman (26 Tests)
6. Ned Hanigan (15 Tests)
7. Michael Hooper (c) (85 Tests)
8. David Pocock (73 Tests)
9. Will Genia (94 Tests)
10. Kurtley Beale (78 Tests)
11. Marika Koroibete (15 Tests)
12. Matt Toomua (37 Tests)
13. Reece Hodge (31 Tests)
14. Israel Folau (67 Tests)
15. Dane Haylett-Petty (25 Tests)
16. Brandon Paenga-Amosa (3 Tests)
17. Sekope Kepu (97 Tests)
18. Allan Alaalatoa (27 Tests)
19. Rory Arnold (17 Tests)
20. Rob Simmons (88 Tests)
21. Nick Phipps (67 Tests)
22. Bernard Foley (62 Tests)
23. Jack Maddocks (4 Tests)