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Skeletal muscle growth is largely dependent on the proliferation and differentiation of muscle-specific stem cells known as satellite cells (SC). Previous work has shown that dietary inclusion of the vitamin D3 metabolite, 25-hydroxycholecalciferol (25OHD3), also called calcidiol, can promote skeletal muscle growth in post-hatch broiler chickens. Improving vitamin D status of broiler breeder hens by feeding 25OHD3 in addition to vitamin D3 has also been shown to positively impact progeny. Yet, whether combined pre- and post-hatch supplementation with 25OHD3 produces an additive or synergistic SC-mediated, skeletal muscle growth response remains unanswered. To evaluate the effect of combined maternal and post-hatch dietary 25OHD3 supplementation on the growth and SC mitotic activity of the Pectoralis major (PM) muscles in broiler chickens, a randomized complete block design experiment with the main effects of maternal diet (MDIET) and post-hatch diet (PDIET) arranged in a 2 × 2 factorial treatment structure was conducted. From 25 to 36 wk of age, broiler breeder hens were fed 1 of 2 MDIET formulated to provide 5,000 IU D3 (MCTL) or 2,240 IU of D3 + 2,760 IU of 25OHD3 per kg of feed (M25OHD3). Their male broiler chick offspring (n = 400) hatched from eggs collected from 35 to 36 wk of age were reared in raised floor pens. Broilers were fed 1 of 2 PDIET formulated to provide 5,000 IU of D3 per kg of feed (PCTL) or 2,240 IU of D3 + 2,760 IU of 25OHD3 per kg of feed (P25OHD3). Muscle was collected at days 4, 8, 15, 22, and 29 and stored until immunofluorescence analysis. Data were analyzed as a 2-way ANOVA with SAS GLIMMIX. Dietary 25OHD3 was effectively transferred from hen plasma to egg yolks (P = 0.002) and to broiler progeny plasma (days 4 to 22; P ≤ 0.044). Including 25OHD3 in either MDIET or PDIET altered PM hypertrophic growth prior to day 29 (P ≥ 0.001) and tended to reduce Wooden Breast severity (P ≤ 0.089). Mitotic SC populations were increased in PM of MCTL:P25OHD3 and M25OHD:PCTL-fed broilers at d 4 (P = 0.037). At d 8, the PM mitotic SC populations were increased 33% by P25OHD3 (P = 0.054). The results of this study reveal that combined maternal and post-hatch 25OHD3 supplementation does not produce additive or synergistic effects on SC-mediated broiler muscle growth. However, vitamin D status improvement through dietary 25OHD3 inclusion in either the maternal or post-hatch diet stimulated broiler breast muscle growth by increasing proliferating SC populations., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)