Open AccessGroupers are an important mariculture in Taiwan and Southeast Asian countries. The rapidly growing orange spotted grouper Epinephelus coioides has experienced relatively severe bacterial disease problems. The proliferation of pathogens in fish can be suppressed by commensal microbe. In this research, probiotic seem to offer an attractive alternative. Bacillus subtilis is a probiotic bacterium that is administered in diet to suppress proliferation of pathogens. In the present study, E. coioides were fed for 6 months with diets contain B. subtilis. Percent weight gain and feed efficiency of the fish fed diets containing 0.1% or 1% probiotics (w/w) were significantly better than the control. The innate cellular response, respiratory burst of the supplemental groups was significantly higher compared to the control on 10 or 20 days after feeding, and even more significant on 30 days. Probiotic B. subtilis increased the intestinal microbial diversity by stimulating the bacterial populations of Paenibacillus sp, Lactobacillus oeni strain 59 b, and Methilacidophilum infernorum strain V4 that are beneficial for E. coioides. The best dose of probiotic B. subtilis based on growth performances, innate cellular responses and microbial profile of fish intestines is 0.1%, which showed equal efficacy as the 1% diet.