Magnesium (Mg) is essential to skeletal muscle where it plays a key role in myofiber relaxation. Although the importance of Mg in the mature skeletal muscle is well established, little is known about the role of Mg in myogenesis. We studied the effects of low and high extracellular Mg in C2C12 myogenic differentiation. Non-physiological Mg concentrations induce oxidative stress in myoblasts. The increase of reactive oxygen species, which occurs during the early phase of the differentiation process, inhibits myoblast membrane fusion, thus impairing myogenesis. Therefore, correct Mg homeostasis, also maintained through a correct dietary intake, is essential to assure the regenerative capacity of skeletal muscle fibers.