Khama handed power to his then-deputy Mokgweetsi Masisi last year after serving as president of the diamond-rich southern African nation for a decade, and he remains an influential figure in the Botswana Democratic Party (BDP).
But Masisi, only the third person from outside the Khama political dynasty to lead Botswana since independence from Britain, has clashed repeatedly with his former ally since he took over.
Their latest disagreement was over Masisi's decision to lift the suspension on big game hunting imposed by Khama's government in 2014.
Khama told a gathering on Saturday in the northeastern village of Serowe, where he is paramount chief, he was switching support from the BDP to opposition alliance Umbrella for Democratic Change (UDC) ahead of an October general election.
"I came here to tell you that I am cutting ties with the BDP as I do not recognise this party anymore. It was a mistake to choose Masisi as my successor. I will now work with the opposition to make sure that the BDP loses power in October," Khama said.
Khama served the maximum two terms as president before stepping down in a scripted succession that compelled him to hand power to his deputy.
In a 2014 general election, the BDP failed for the first time to score an outright majority as the country struggles to make its ailing economy less reliant on diamond sales.