Abstract: To investigate the effects of Bacillus subtilis as water additive on gut microbiota in Spinibarbus caldwelli,Acrossheilus hemispinus and Hypophthalmichthys molitrix and its purification effect on aquaculture water,the gut microflora in the foregut,midgut and hindgut of three fishes were investigated through 16S rDNA sequencing and the pH,the levels of ammonia nitrogen and nitrite in the aquatic water were measured.The results suggested B.subtilis could reduce the contents of ammonia nitrogen by 46.39% in water stably since the 11th day and nitrite by 84.61% in water stably since the 19th day.The gut microflora in each intestinal site between experiment and control group were significantly different overall and the abundance of intestinal flora decreased in the experiment group.On the whole,in experiment group,the abundance of intestinal microbiota decreased and the phylum abundance of Actinobacteria and Planctomycetes increased and decreased,respectively.The genus levels of Bacillus increased while pathogenic bacteria Aeromonas and Vibrio decreased.The KEGG signal pathways showed that the metabolic functions of the H.molitrix were enhanced.This study showed that B.subtilis could reduce the level of ammonia nitrogen and nitritein in the aquatic water and purify the aquatic wate,while inhibiting the propagation of harmful intestinal microbiota and improving the metabolic level of intestinal microbiota in H.molitrix.