Four hundred and eighty five rainbow trout (76 ± 6.44 g mean weight) were acclimated to laboratory conditions and then randomly divided into four groups of tanks in triplicate. The first group (Group 1) was fed on a commercial diet (control) without Bacillus subtilis supplementation. The second, third and fourth groups (Group 2, Group 3 and Group 4, respectively) were given a diet supplemented with 1, 5 and 10 g probiotic powder (containing 8×10⁷ CFU g^-1Bacillus subtilis) per kg commercial feed. Growth performance, immune responses and glucose levels were analyzed on days 0, 22 and 44. The results showed that dietary Bacillus subtilis supplementation significantly (p<0.05) reduced final weight in treated groups compared with that in the control group. No significant difference (p> 0.05) was observed in weight gain rate (WGR) between the control and group 2. On the effect of dietary Bacillus subtilis on serum lysozyme, it was observed that group 3 and group 4 did not show any significant differences in serum lysozyme activity and serum total antibody on day 44. Results of alternative complement activity, showed significant increase during the experimental days (p< 0.05). Results on glucose assay showed that group 3 had the lowest glucose level (13.71 mg/dL) which was not significantly different than that in other groups on day 44. Fish diet supplementation with 5 g probiotic powder (Group 3) is preferable for immune system responses; however, high dose of Bacillus subtilis may be helpful to improve growth performance in rainbow trout,Oncorhynchus mykiss (Walbaum, 1792).