The ciliate Balantidium ctenopharyngodoni is the most prominent protist in the guts of grass carp, where it mainly inhabits the creamy luminal contents of the hindgut. Ciliates are generally colonized by microorganisms via phagotrophic feeding. In order to study the intracellular bacteria in this ciliate, we have successfully established it in in vitro culture. Herein, we investigated and compared the bacterial community structures of cultured and freshly collected B. ctenopharyngodoni. The results showed that these two groups exhibited different bacterial communities. The most abundant bacterial family in freshly collected samples was Enterobacteriaceae, while in cultured samples it was Fusobacteriaceae. In addition, a key intracellular bacterium, Cetobacterium somerae, was identified in the cytoplasm of cultured ciliates using fluorescence in situ hybridization (FISH). This study shows that ciliates can retain the intracellular bacteria acquired in the natural habitat for quite a long time, but the bacterial community structure of ciliates eventually changes after a long period of cultivation.