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. In addition to the effects on T3SS function, the absence of the chaperone BP2265 results in increased production of numerous flagellar genes and several virulence genes. The increased production of flagellar proteins and intimin BipA translated into increased biofilm formation by the mutant.