England international Sam, 24, Luke, 26, and 21-year-old twins George and Tom - weighing in at a combined 460 kg (1,014 pounds) - have all been named in the squad for Friday's match against West Tigers at the Sydney Football Stadium (SFS).
It will be the first time in 103 years that four brothers have played together at the top level of rugby league, the number one winter sports code on the east coast of Australia.
The Rabbitohs, co-owned by Hollywood actor Russell Crowe, are one of Australia's oldest rugby league teams but won the last of their 20 titles in 1971.
Level with points at the top of the National Rugby League (NRL) standings with the Sydney Roosters, the Rabbitohs will almost certainly need a win on Friday to keep alive their hopes of claiming top spot and home advantage throughout the playoffs.
With a suspension for Sam and an injury to Luke having prevented the four brothers from turning out together before, there is a good chance the four Yorkshiremen will also feature in next week's regular season finale against the Roosters.
"To see them all work extremely hard, it's obviously something as a group they've been really keen to achieve," Rabbitohs coach Michael Maguire told reporters on Wednesday.
"And it's through merit, they thoroughly deserve their positions. Everyone is looking forward to a game like this with four brothers."