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Simple Summary: The excessive or improper use of antibiotics in chicken feed has led to the emergence of antibiotic-resistant bacteria, drug residues in the tissue, and other relevant issues, and this situation is severe in China. From 31 December 2020, antibiotics were banned for use as supplemental growth promoters in animal feed in China; therefore, antibiotic substitutes are urgently needed in China. People are searching for ideal antibiotic alternatives, and probiotics have proven their potential use for this. Our current trial aims to evaluate the effects of the Bacillus subtilis (B. subtilis) YW1 strain on laying hens, including the effects on performance, morphology, cecal microbiota, and intestinal barrier function. Our study showed that orally administering laying hens with probiotic B. subtilis cannot significantly improve their overall egg production, can induce a healthier microbiota composition characterized by a higher ratio of beneficial bacteria, and strengthens the physical barrier function of the intestine by inducing a higher expression of the tight junction protein. This makes it a valid probiotic alternative to antibiotics, and also a reference strain for clinical application in the poultry industry. We investigated the efficacy of a single bacterium strain, Bacillus subtilis (B. subtilis) 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 B. subtilis at a dose rate of 5 × 108 CFU/kg and 2.5 × 109 CFU/kg, respectively. Dietary supplementation with B. subtilis did not significantly affect overall egg production in both groups, with no obvious changes in average egg weight and intestine morphology. B. subtilis administration also improved the physical barrier function of the intestine by inducing significantly greater expression levels of the tight junction protein occludin in T1 (p = 0.07) and T2 (p < 0.05). Further, supplementation with B. subtilis effectively modulated the cecal microbiota, increasing the relative level of beneficial bacteria at the genus level (e.g., Bifidobacterium p < 0.05, Lactobacillus p = 0.298, Bacillus p = 0.550) and decreasing the level of potential pathogens (e.g., Fusobacterium p < 0.05, Staphylococcus p < 0.05, Campylobacter p = 0.298). Overall, B. subtilis 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 B. subtilis YW1 a good probiotic candidate for application in the poultry industry, and further expanding the resources of strains of animal probiotics. [ABSTRACT FROM AUTHOR]