Simple SummaryDue to breeding for high egg production, laying hens are at great risk for developing osteoporosis. To develop an effective feed additive for reducing the bone damage and associated pain and economic loss has become a critical issue affecting the poultry industry. The aim of this study was to investigate the effects of Bacillus subtills as a feed supplement on production performance and bone pathophysiological characteristics of laying hens. The results showed that Bacillus subtilis increases marketable eggs, protects bone health, changes the distribution of phosphorus between blood and bone, and increases estrogen but decreases interleukin-1 and tumor necrosis factor-α concentrations in blood. Results indicate that Bacillus subtilis can be used as a dietary supplement to increase marketable egg production and bone health of laying hens by inhibiting gut and systemic inflammation via the microbiota-gut-immune and the microbiota-gut-bone axes.This study was to investigate the effects of Bacillus subtilis on production performance and bone pathophysiological characteristics of layers. Twenty-four 48-week-old Lohmann Pink-shell laying hens were randomly divided into two groups: a basic diet (control) and the basic diet mixed with Bacillus subtilis (0.5 g/kg) for a 60-day trial. Statistically, independent-sample t-test was used to assess the treatment differences. The results showed that Bacillus subtilis supplementation improved the percent of marketable eggs (p < 0.05) with reduced numbers of broken and soft-shelled eggs but had no effects on egg weight, height of albumen, yolk color, and Haugh unit (p > 0.05). Bacillus subtilis supplement also elevated maximum load (p = 0.06), maximum stress (p = 0.01), stiffness (p < 0.01), and Young’s modulus (p < 0.01) but suppressed maximum strain (p = 0.06) in the femur. In addition, compared with control birds, phosphorous concentration (p < 0.01) was reduced in serum at day 61 but increased in the femur (p < 0.05) in Bacillus subtilis fed birds. Bacillus subtilis fed birds also had lower magnesium concentrations in both femur (p = 0.04) and feces (p = 0.09). Furthermore, Bacillus subtilis increased plasma estrogen concentration (p = 0.01) and femur TNF receptor superfamily member 11b (OPG) expression (p < 0.05) but reduced plasma IL-1 (p < 0.01) and TNF-α (p < 0.01) concentrations. These results indicate that Bacillus subtilis could be used as a health promotor to reduce overproduction-induced inflammation and associated bone damage and to increase marketable egg production. The data provide evidence for developing a management strategy to use Bacillus subtilis as a feed additive to improve marketable egg production and health and welfare status of laying hens.