Bordetella pertussis is a Gram-negative, strictly human respiratory pathogen and the causative agent of whooping cough (pertussis). Similar to other Gram-negative pathogens, B. pertussis produces a functional type III secretion system, but its role in pathogenesis of B. pertussis is enigmatic and has not yet been elucidated. Here, we applied omics RNA-seq as well as LC-MS/MS techniques and co-immunoprecipitation method to identify and characterize the novel CesT family T3SS chaperone BP2265. Our results show that the chaperone BP2265 specifically interacts with the secreted T3SS anti-sigma factor BtrA. Moreover, in the absence of the chaperone, secretion but not production of BtrA and several early, intermediate, and late T3SS substrates is severely impaired. It appears that the role of BtrA in regulating T3SS is more complex and extends beyond its activity as an antagonist of the sigma factor BtrS. We propose to rename BP2265 as BtcB for the Bordetella type III chaperone of BtrA.