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Probiotics are considered ecofriendly alternatives to antibiotics as immunostimulants against pathogen infections in aquaculture. In the present study, protease-, amylase-, cellulase-, and xylanase-producing <i>Bacillus safensis</i> NPUST1 were isolated from the gut of Nile tilapia, and the beneficial effects of <i>B. safensis</i> NPUST1 on growth, innate immunity, disease resistance and gut microbiota in Nile tilapia were evaluated by feeding tilapia a basal diet or basal diet containing 10⁵ and 10⁶–10⁷ CFU/g for 8 weeks. The results showed that the weight gain, feed efficiency and specific growth rate were significantly increased in tilapia fed a diet containing 10⁶ CFU/g and 10⁷ CFU/g <i>B. safensis</i> NPUST1. Intestinal digestive enzymes, including protease, amylase and lipase, and hepatic mRNA expression of glucose metabolism and growth-related genes, such as <i>GK</i>, <i>G6Pase</i>, <i>GHR</i> and <i>IGF-1</i>, were also significantly increased in the 10⁶ CFU/g and 10⁷ CFU/g <i>B. safensis</i> NPUST1 treated groups. Immune parameters such as phagocytic activity, respiratory burst and superoxide dismutase activity in head kidney leukocytes, serum lysozyme, and the mRNA expression of <i>IL-1β</i>, <i>IL-8</i>, <i>TNF-α</i> and <i>lysozyme</i> genes were significantly induced in the head kidney and spleen of 10⁶ CFU/g and 10⁷ CFU/g <i>B. safensis</i> NPUST1 treated fish. The cumulative survival rate was significantly increased in fish fed a diet containing 10⁶ CFU/g and 10⁷ CFU/g <i>B. safensis</i> NPUST1 after challenge with <i>Streptococcus iniae</i>. Dietary supplementation with <i>B. safensis</i> NPUST1 improves the gut microbiota of Nile tilapia, which increases the abundance of potential probiotics and reduces the abundance of pathogenic pathogens. The present study is the first to report the use of <i>B. safensis</i> as a potential probiotic in aquaculture, and a diet containing 10⁶ CFU/g <i>B. safensis</i> NPUST1 is adequate for providing beneficial effects on growth performance and health status in tilapia.