The probiotic properties of Bacillus subtilis HAINUP40 isolated from the aquatic environment, and the effects of dietary administration of B.subtilis HAINUP40 on the growth performance, intestinal probiotic recovery, digestive enzyme activities, innate immunity and disease resistance of tilapia (Oreochromis niloticus) were evaluated.The probiotic properties investigated include tolerance to simulated gastrointestinal stress, auto-aggregation, cell surface hydrophobicity and extracellular enzyme production.The cell number ofB.subtilis changed little after 4 h in simulated gastric fluid at pH =2.0,3.0, 4.0 and small intestinal fluid at pH =6.8.B.subtilis HAINUP40 revealed a strong auto-aggregation property (34.6-87.0%) after a 24h incubation period.It exhibited significant cell surface hydrophobicity in xylene (28.8%) and chloroform (41.3%) and produced extracellular proteases and amylase.After tilapia (mean weight =95.02 ± 7.65 g) were fed with a diet containing 108cfu/g B.subtilis HAINUP40,their final body weight, percent weight gain (PWG), specific growth rate (SGR), total antioxidant capacity (T-AOC) and serum superoxide dismutase (SOD) increased significantly (p<0.05) after 8 weeks;feed conversion rate (FCR) is significantly lower (p < 0.05) after 8 weeks;the protease and amylase activity in the digestive tract increased significantly (p<0.05) after 4 and 8 weeks;and respiratory bursts and serum lysozyme activity increased significantly (p< 0.05) after 2 weeks.Moreover,being challenged with pathogenic Streptococcus agalactiae for 2 weeks, the relative percent survival (RPS%) is 52.94%.The results of this study strongly suggest that dietary supplement of B.subtilis HAINUP40 can effectively enhances the growth performance, immune response, and disease resistance of Nile tilapia.