Vitamin D deficiency is linked to a range of muscle disorders including myalgia, muscle weakness, and falls. Humans with severe vitamin D deficiency and mice with transgenic vitamin D receptor (VDR) ablation have muscle fiber atrophy. However, molecular mechanisms by which vitamin D influences muscle function and fiber size remain unclear. A central question is whether VDR is expressed in skeletal muscle and is able to regulate transcription at this site. To address this, we examined key molecular and morphologic changes in C2C12 cells treated with 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25(OH)(2)D). As well as stimulating VDR expression, 25(OH)D and 1,25(OH)(2)D dose-dependently increased expression of the classic vitamin D target cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1), demonstrating the presence of an autoregulatory vitamin D-endocrine system in these cells. Luciferase reporter studies demonstrated that cytochrome P450, family 27, subfamily B, polypeptide 1 (CYP27B1) was functional in these cells. Both 25OHD and 1,25(OH)(2)D altered C2C12 proliferation and differentiation. These effects were related to the increased expression of genes involved in G(0)/G(1) arrest (retinoblastoma protein [Rb], 1.3-fold; ATM, 1.5-fold, both P < .05), downregulation of mRNAs involved in G(1)/S transition, including myc and cyclin-D1 (0.7- and 0.8-fold, both P < .05) and reduced phosphorylation of Rb protein (0.3-fold, P < .005). After serum depletion, 1,25(OH)(2)D (100nM) suppressed myotube formation with decreased mRNAs for key myogenic regulatory factors (myogenin, 0.5-fold; myf5, 0.4-fold, P < .005) but led to a 1.8-fold increase in cross-sectional size of individual myotubes associated with markedly decreased myostatin expression (0.2-fold, P < .005). These data show that vitamin D signaling alters gene expression in C2C12 cells, with effects on proliferation, differentiation, and myotube size.