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We investigated the efficacy of a single bacterium strain, <i>Bacillus subtilis</i> (<i>B. subtilis</i>) YW1, on the performance, morphology, cecal microbiota, and intestinal barrier function of laying hens. A total of 216 28-week-old Hy-line Brown laying hens were divided into three dietary treatment groups, with six replicates of 12 birds each for 4 weeks. The control group (Ctr) was fed a basal diet and the treatment groups, T1 and T2, were fed a basal diet supplemented with <i>B. subtilis</i> at a dose rate of 5 × 10⁸ CFU/kg and 2.5 × 10⁹ CFU/kg, respectively. Dietary supplementation with <i>B. subtilis</i> did not significantly affect overall egg production in both groups, with no obvious changes in average egg weight and intestine morphology. <i>B. subtilis</i> administration also improved the physical barrier function of the intestine by inducing significantly greater expression levels of the tight junction protein occludin in T1 (<i>p</i> = 0.07) and T2 (<i>p</i> < 0.05). Further, supplementation with <i>B. subtilis</i> effectively modulated the cecal microbiota, increasing the relative level of beneficial bacteria at the genus level (e.g., <i>Bifidobacterium</i> <i>p</i> < 0.05, <i>Lactobacillus p</i> = 0.298, <i>Bacillus</i> <i>p</i> = 0.550) and decreasing the level of potential pathogens (e.g., <i>Fusobacterium p</i> < 0.05, <i>Staphylococcus p</i> < 0.05, <i>Campylobacter p</i> = 0.298). Overall, <i>B. subtilis</i> YW1 supplementation cannot significantly improve the egg production; however, it modulated the cecal microbiota towards a healthier pattern and promoted the mRNA expression of the tight junction protein occludin in laying hens, making <i>B. subtilis</i> YW1 a good probiotic candidate for application in the poultry industry, and further expanding the resources of strains of animal probiotics.