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The study investigated the effects of dietary probiotic of dual-strain Bacillus subtilis (BS) on production performance, intestinal barrier parameters, and microbiota in broiler chickens. In a randomized trial, male broiler chickens were allocated into three groups, a control group (basal diet), BS300 group (basal diet with 300 mg/kg BS), and BS500 group (basal diet with 500 mg/kg BS). The inclusion of 500 mg/kg BS significantly reduced the feed conversion ratio by 4.55% during the starting phase. Both 300 and 500 mg/kg BS supplementation increased jejunal villus height (by 17.89% and 24.8%, respectively) significantly and decreased jejunal crypt depth (by 27.2% and 31.9%, respectively) on day 21. The addition of 500 mg/kg BS significantly elevated the gene expression of occludin on day 35. Moreover, BS supplementation enhanced cytokine levels and immunoglobulins in both serum and jejunal mucosa. Microbial analysis indicated that BS increased the abundance of potential probiotics (Sutterella) and butyrate-producing bacteria (Lachnoclostridium, Tyzzerella, Anaerostipes, Clostridium_sensu_stricto_13, Prevotellaceae_NK3B31_group, and Lachnospiraceae_UCG-010). The abundances of Anaerostipes and Sutterella, are significantly correlated with growth performance and immune function. In conclusion, dietary supplementation with BS improved the growth performance, potentially through the regulation of immunity, intestinal barrier function, and microbiota in broilers. Notably, 500 mg/kg of BS exhibited more benefits for broilers compared to the 300 mg/kg., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)