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The expanded tournament will be called the Pro14 and see the teams split into two conferences of seven made up of two Welsh, two Irish and one team each from Scotland, Italy and South Africa.
The conferences have been decided based on last season's results.
Cheetahs and Kings will play their home games in South Africa.
They will play only Saturdays, and there will be five travel-free days before matches.
The first round of matches is due to be played on the weekend of 1-3 September, with the first fixtures to be announced on 7 August.
It is understood the addition of the two new teams will bring in an extra £6m a year in revenue.
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"The arrival of the Toyota Cheetahs and the Southern Kings marks a bold and exciting new chapter for the Guinness PRO14 as a global rugby Championship," said Pro14 chief executive Martin Anayi.
"South Africa is a rugby powerhouse of over 55 million people. These teams already operate to the high standards demanded by Super Rugby and they will add to the quality of our tournament.
"This is a natural evolution for the Championship... and we aim to be at the forefront of the game's growth around the world."
The South African teams lost their places in the southern hemisphere Super Rugby, which is cutting from 18 teams to 15, at the end of last season.
The Pro12 grew out of the Celtic League, which was formed in 2001 but has seen many changes to participants and format during its existence.
Last season's tournament was won by Scarlets, who beat Munster in the play-off final in Dublin.
Jurie Roux, CEO of SA Rugby, says the decision for South African team to play in a northern hemisphere tournament was groundbreaking.
"This development is as exciting as the launch of Super Rugby itself back in 1996," he said.
"It will not be without its challenges in aligning with a competition in a different part of the calendar and in very different playing conditions; but it is also a fantastic opportunity for South African rugby to widen our rugby horizons.
"We believe the Toyota Cheetahs' and Kings' participation will be good for the competition and good for the teams."
The Pro12 will be expanded to include South African side Southern Kings and Cheetahs from September, organisers have confirmed.