Symbionts are associated with many insects and play several multifunctional roles in insect-microorganism mutualistic relationships. The trichlorphon-degrading symbiont Citrobacter freundii (CF-BD) of the oriental fruit fly Bactrocera dorsalis was recently discovered; however, its intraspecies transmission pathway among flies remains unknown. Here, we use fluorescence in situ hybridization (FISH), PCR detection, and a series of ingenious experiments to reveal that CF-BD was aggregated in rectal pads associated with the female ovipositor, and the CF-BD symbiont was vertically transmitted via egg surface contamination. Although CF-BD was not detected in ovaries, it was found in deposited eggs. In addition, CF-BD was readily acquired horizontally between larvae or adults via oral uptake, although it was not transferred via mating behavior. Surface sterilization of eggs had a negative effect on the insects, which exhibited a lower body weight and a sharp decrease in fecundity, suggesting important biological roles of CF-BD in the fitness of the host insects. Our findings may also help to explain the high pesticide resistance levels of B. dorsalis. Furthermore, identifying a clear transmission pathway of this organophosphorus-degrading symbiont will be useful for pesticide resistance management and future pest control technologies.