The active form of Vitamin D (1,25(OH)2D), has been suggested to have a regulatory role in skeletal muscle function and metabolism, however, the effects and mechanisms of vitamin D (VitD) action in this tissue remain to be fully established. In this study, we have used primary human skeletal muscle myoblast (HSMM) cells that display typical characteristics of human skeletal muscle function and protein levels, to investigate the effects of the active form of VitD on proliferation, differentiation, protein synthesis and bioenergetics. Myoblast cells were treated with 100 nM of VitD for 24 h, 48 h, 72 h and five days (cells were differentiated into myotubes) and then analyses were performed. We report that VitD inhibits myoblast proliferation and enhances differentiation by altering the expression of myogenic regulatory factors. In addition, we found that protein synthesis signaling improved in myotubes after VitD treatment in the presence of insulin. We also report an increase in oxygen consumption rate after 24 h of treatment in myoblasts and after 5 days of treatment in myotubes after VitD exposure. VitD significantly impacted HSMM myogenesis, as well as protein synthesis in the presence of insulin.