Celtic Rugby and the South African Rugby Union have been in talks for several weeks about including the southern hemisphere club sides in what was formally the Pro12.
The Southern Kings and the Cheetahs were in need of a new home after being culled from Super Rugby and will now take on the best Wales, Scotland, Ireland and Italy has to offer.
Organisers have announced the competition will be split into two conferences of seven featuring two Welsh, two Irish and one team each from Scotland, Italy and South Africa, with all sides playing 21 matches in the regular season.
The Kings and Cheetahs will continue to play their home games in South Africa, with their matches only taking place on a Saturday and sides having five travel-free days before games.
It is understood the addition of the two new teams will bring in an extra £6million in revenue and has been hailed as the start of a new global era for club rugby by Pro14 chief executive Martin Anayi.
"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 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 first round of matches are due to take place between September 1-3, with the fixtures announced on August 7.