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Probiotics as dietary additives can improve weight gain, feed efficiency, and disease resistance in cultured fish. In this research, we evaluated and compared the effects of Bacillus subtilis on immunity, mucosal tissue morphology, immune-related gene transcriptions, and intestinal microbiota in flounder (Paralichthys olivaceus) by a 30-day feeding experiment based on a continuous feeding schedule (E1) and a discontinuous feeding schedule (E2). As a result, the use of B. subtilis exerted the best positive effects on survival rate, enzyme activity, mucosal tissue morphology, immune-related gene transcriptions, and intestinal microbiota in flounders. Alkaline phosphatase (AKP), lysozyme (LZM), and superoxide dismutase (SOD) activities in the liver of E2 were higher than those of E1 (P < 0.05). Furthermore, the villi length in the intestinal tract and the fold length in the stomach of E2 were also higher than in E1 (P < 0.05). The il-1 expression levels in the spleen were significantly increased in E2 (P < 0.05) compared to E1. We performed 16 S rRNA sequencing analysis to find that Bacillus in E1 (1.06%) and E2 (1.01%) had higher relative abundances than in E0 (0.053%) at the end of the experiments, indicating that short-term application of B. subtilis with the continuous or discontinuous feeding method can allow both the adaptation of the ecosystem to the presence of probiotics by the establishment of new species in the gut microbiota and the ability these new probiotic species to perform corresponding functions. No significant differences in the ability of probiotic establishment were observed between E1 and E2. Our findings provided a unique perspective to explore the mechanism of immune enhancement with probiotics and to screen the optimal administration strategy in aquaculture application for probiotic use. Together, these results point to some level of enhancement in immune status by continuous and discontinuous feeding after a short-term feeding period, which could be used as a prophylactic strategy for flounder health management. [ABSTRACT FROM AUTHOR]