In this study, the effects of dietary probiotic Bacillus subtilis C-3102 (Calsporin®) on the growth performance, predominant intestinal microbiota, expression of cytokines genes in three organs (liver, intestine and kidney) and protection against Aeromonas hydrophila infection of koi carp were investigated. Fish were fed two different diets, 1-control diet (non-supplemented) and 2-experimental diet (supplemented with 1 g/kg Calsporin®) for five weeks. Tissue samples were collected at days 0, 10, 20 and 35. The results showed that the weight gain (WG) and feed conversion ratio (FCR) were significantly improved by dietary Bacillus subtilis C-3102 (P < 0.01). Dietary probiotic did not affect the body skin colouration of koi carp (P > 0.05). Variation of intestinal bacterial communities were studied by denaturing gradient gel electrophoresis (DGGE) which revealed that even though Bacillus subtilis C-3102 was not detected as a prominent component in the intestinal tract of koi carp at any time point, it could affect the intestinal microbiota community at the early stages of the trial, becoming weaker in the later stages. Concerning the gene expression results, the expression of HSP70 gene was up-regulated at day 10 and 35 the liver; no effects were observed in the intestine and kidney. A general trend of upregulation of cytokines expression (IL-1β, IL-10, TNF-α and TGF-β) was observed in liver the (except IL-1β) and intestine, but unchanged in the kidney (except IL-10). The intraperitoneal injection challenge demonstrated that there was no positive effect of dietary B. subtilis C-3102 supplementation against A. hydrophila. These results suggested that B. subtilis C-3102 can provide beneficial effects on growth, feed utilization and modulating intestinal microbiota community.