Tom Koutsantonis has been appointed treasurer and Attorney-General Kyam Maher is the deputy premier after being elected deputy Labor leader by the party's caucus.
They were sworn in at a ceremony at Government House on Friday, along with two other ministers who have been elevated to Cabinet roles.
They replace Susan Close and Stephen Mullighan as deputy premier and treasurer, after Premier Peter Malinauskas announced on Thursday the pair had indicated they would retire at the March 2026 state election.
Adelaide MP Lucy Hood replaces Dr Close as environment minister, and King MP Rhiannon Pearce takes on emergency services and correctional services as well as recreation, sport and racing.
Mr Malinauskas said the reshuffle brought "significant new talent and fresh ideas to the front bench, balanced with stability, experience, continuity and strength of leadership".
"I am proud of the fact that for the first time in history we have more women than men in Cabinet," he said.
Mr Koutsantonis previously served as treasurer in then premier Jay Weatherill's government from 2014-18.
Ms Hood will take on the challenging role of managing the government's response to the state's algal bloom, which has devastated marine life and led to residents in coastal regions reporting flu-like symptoms since it was detected in March.
Opposition deputy leader Josh Teague said the government was "seemingly entirely preoccupied with a crisis management announcement … and then appeared to pat itself on the back at its capacity to spin this crisis at the top levels of government".
"Meanwhile, the substantial day-to-day tasks of the work of government have gone very much neglected this week, and that's particularly true of the state's coastal ecological disaster, the ongoing harmful algal bloom," he said.
"The government's response to the algal bloom crisis has been flat-footed from the very beginning and the premier has just hospital-passed this bungle to a first-time minister."
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