Skeletal muscle wasting is an important public health problem associated with aging, chronic disease, cancer, kidney dialysis, and HIV/AIDS. 1,25-Dihydroxyvitamin D (1,25-D3), the active form of vitamin D, is widely recognized for its regulation of calcium and phosphate homeostasis in relation to bone development and maintenance and for its calcemic effects on target organs, such as intestine, kidney, and parathyroid glands. Emerging evidence has shown that vitamin D administration improves muscle performance and reduces falls in vitamin D-deficient older adults. However, little is known of the underlying mechanism or the role 1,25-D3 plays in promoting myogenic differentiation at the cellular and/or molecular level. In this study, we examined the effect of 1,25-D3 on myoblast cell proliferation, progression, and differentiation into myotubes. C(2)C(12) myoblasts were treated with 1,25-D3 or placebo for 1, 3, 4, 7, and 10 d. Vitamin D receptor expression was analyzed by quantitative RT-PCR, Western blottings and immunofluorescence. Expression of muscle lineage, pro- and antimyogenic, and proliferation markers was assessed by immunocytochemistry, PCR arrays, quantitative RT-PCR, and Western blottings. Addition of 1,25-D3 to C(2)C(12) myoblasts 1) increased expression and nuclear translocation of the vitamin D receptor, 2) decreased cell proliferation, 3) decreased IGF-I expression, and 4) promoted myogenic differentiation by increasing IGF-II and follistatin expression and decreasing the expression of myostatin, the only known negative regulator of muscle mass, without changing growth differentiation factor 11 expression. This study identifies key vitamin D-related molecular pathways for muscle regulation and supports the rationale for vitamin D intervention studies in select muscle disorder conditions.