This study was conducted to evaluate the effects of Bacillus subtilis SC02 supplement on water quality, microbial community diversity and structure in a grass carp (Ctenopharyngodon idellus) culture. Our selected strain, B. subtilis SC02, significantly reduced ammonia, nitrite and total nitrogen levels in water over an extended period compared with the control group. Pyrosequencing showed that the Shannon diversity index (Shannon) and species richness estimators (Chao) of the treatment group were higher, indicating that bacterial richness was significantly increased in the treatment group. The phyla Proteobacteria, Bacteroidetes and Actinobacteria were dominant in the treatment groups, accounting for 45, 21.9 and 21.9 % of the sequence reads, respectively. However, in sharp contrast, the control fishes were predominantly occupied by Proteobacteria (82.1 %) and Firmicutes (8.4 %). At the genus level, the microbial communities were different between the control and treatment groups, although the two groups shared similar genera. Additionally, some genera (such as Tepidimonas, Variovorax, Roseomonas, Rubritepida, Nitrobacter, etc.) only appeared in the treatment group, and many other genera only existed in the control group. Therefore, we conclude that the addition of the SC02 strain in water improves water quality, which may ultimately be a result of changes in microbial community diversity in grass carp cultures.