This study investigated the efficacy of a single strain of Bacillus subtilis (SSB) in modulating the composition of cecal microbiota and its link to the concentration of short-chain fatty acids (SCFA) and apparent retention (AR) of components. A total of 720, 4-week-old Shaver White chicks were allotted to control (CON), 1.1Eϩ08 (low, LSSB), 2.2Eϩ08 (medium, MSSB), or 1.1Eϩ09 (high, HSSB) CFU/kg of diet groups. At grower (10-week), developer (16-week), and laying (28-week) phases, excreta and cecal digesta samples were taken for AR, microbial, and SCFA analyses. Microbial analysis involved high-throughput sequencing of the V3-V4 hypervariable regions of 16S rRNA gene. Bacterial diversity decreased (P Ͻ 0.05) at the developer phase as the SSB dose increased; however, a distinct clustering pattern (P Ͻ 0.05) of bacterial community was noted. Bacteroides and Faecalibacterium were differentially enriched in the developer for SSB-fed compared to CON-fed birds. Although no differences in microbial diversity were detected in grower and layer phases, different species of Clostridium (XVIII, XIVa, IV, and XIVb)-major butyrate producers-were identified in all phases, with stronger effect sizes for SSB-fed compared to CON-fed birds. Isobutyric acid was elevated in dose response (P ϭ 0.034) in layer phase. In addition, the relative abundances of Alistipes, Lactobacillus, and Bifidobacterium were positively correlated (P Ͻ 0.05), with AR of most components for SSB-fed birds in the pullet phase. The results suggested that supplementing chickens' diet with B. subtilis DSM 29784 may selectively enrich beneficial bacterial communities, which in turn are critical in promoting the growth and performance of hens. IMPORTANCE In egg-laying chickens, the trend in the move away from the cage to alternative housing systems and restriction in antimicrobial use requires alternative approaches to maintain health and prevent diseases. There is increased research and commercial interest toward alternative gut health solutions while improving the performance and product safety in poultry production systems. One such approach, in recognition of the importance of the gut microbial community, is the use of microbes as feed supplements (such as probiotics). Unlike meat-type chickens, studies assessing the efficacy of such microbial supplements are limited for egg-laying chickens. Thus, by conducting a comprehensive assessment of the hen microbiota in response to various levels of B. subtilis DSM 29784 during the pullet phase (grower and developer) and the layer phase, the present study demonstrates the importance of direct-fed microbes in modulating gut microbiome, which may relate to improved performance efficiency in the pullet and layer phases.Citation Neijat M, Habtewold J, Shirley RB, Welsher A, Barton J, Thiery P, Kiarie E. 2019. Bacillus subtilis strain DSM 29784 modulates the cecal microbiome, concentration of shortchain fatty acids, and apparent retention of dietary components in Shaver White chickens during grower, developer, and laying ...