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The effects of dietary administration of Bacillus subtilis on the growth, digestive enzyme activity, blood chemistry, oxidative status and immune response of red sea bream (Pagrus major) were evaluated in the current study. Fish fed five different levels of B. subtilis at 0 (BS0), 1×104 (BS1), 1×106 (BS2), 1×108 (BS3) and 1×1010 (BS4) CFU kg−1 diet for 60 days. The obtained results showed that B. subtilis supplementation significantly improved growth performance (FBW, WG and SGR), feed utilization (FI, FCE, PER and PG) and whole-body protein content when compared to the control group (P<0.05). Furthermore, the specific activities of amylase, protease and lipase enzymes up regulated significantly upon B. subtilis incorporation in red sea bream diets (P<0.05). No changes have been reported on blood biochemical variables except for the plasma total protein, which increased significantly in fish fed BS3 diet when compared with the control diet (P<0.05). Hematocrit, hemoglobin and the nitro blue tetrazolium values also reported the highest values significantly in fish fed B. subtilis, especially in case of BS3 and BS4 diets (P<0.05). Serum bactericidal activity enhanced significantly in BS2, BS3 and BS4 groups (P<0.05), while mucus bactericidal activity showed no significant activity among tested groups (P>0.05). Serum lysozyme activity exhibited higher values in case of BS3 and BS4 groups than BS0 group (P<0.05), while mucus lysozyme activity increased only in BS3 group. Also, serum peroxidase activity enhanced significantly in fish fed BS2 and BS3 diets (P<0.05), however, no activities were observed in the collected mucus. All groups reflexed high tolerance ability against oxidative stress except for BS0 and BS1 groups. Additionally, catalase activity increased significantly in all B. subtilis fed groups when compared to BS0 group (P<0.05). Considering the obtained results, the supplementation of B. subtilis in the diet of red sea bream at 1×108 and 1×1010CFUkg−1 diet could improve the growth, feed utilization, health condition and immune response.We studied the effects of Bacillus subtilis on red sea bream (Pagrus major) for 60 days.Administration of B. subtilis can be considered as an effective probiotic for red sea bream.B. subtilis showed the improvement of growth performances and feed utilization of red sea bream after 60 days.B. subtilis up regulated the activity of digestive enzymes.B. subtilis-supplemented diets enhanced the antioxidative status and immune responses of red sea bream.