Ectopic calcification occurs in the skeletal muscle of mdx mice, a dystrophin-deficient animal model of Duchenne muscular dystrophy. The purpose of this study was to clarify the mechanism of the calcification. The calcified deposits were identified as hydroxyapatite, a crystallized form of calcium phosphate, and the serum inorganic phosphate (Pi) level in the mdx mice was approximately 1.4 times higher than that in the normal B10 mice, suggesting that Pi plays a critical role in the ectopic calcification. When C2C12 mouse myoblasts were cultured under high-Pi conditions, myogenic differentiation was retarded while the expression of osteogenic markers such as osteocalcin and Runx2 were upregulated. This was followed by the generation of calcium deposition. Moreover, ectopic calcification reduced to an undetectable level in most of the mdx mice fed a Pi-reduced diet. We therefore conclude that the Pi-induced osteogenesis of muscle cells is responsible for ectopic calcification in the skeletal muscle of mdx mice.