Sortilin has been implicated in the formation of insulin-responsive GLUT4 storage vesicles in adipocytes by regulating sorting events between the trans-Golgi-network and endosomes. We herein show that sortilin serves as a potent myogenic differentiation stimulator for C2C12 myocytes by cooperatively functioning with p75NTR, which subsequently further contributes to development of the insulin-responsive glucose transport system in C2C12 myotubes. Sortilin expression was up-regulated upon C2C12 differentiation, and overexpression of sortilin in C2C12 cells significantly stimulated myogenic differentiation, a response that was completely abolished by either anti-p75NTR- or anti-nerve growth factor (NGF)-neutralizing antibodies. Importantly, small interference RNA-mediated suppression of endogenous sortilin significantly inhibited C2C12 differentiation, indicating the physiological significance of sortilin expression in the process of myogenesis. Although sortilin overexpression in C2C12 myotubes improved insulin-induced 2-deoxyglucose uptake, as previously reported, this effect apparently resulted from a decrease in the cellular content of GLUT1 and an increase in GLUT4 via differentiation-dependent alterations at both the gene transcriptional and the post-translational level. In addition, cellular contents of Ubc9 and SUMO-modified proteins appeared to be increased by sortilin overexpression. Taken together, these data demonstrate that sortilin is involved not only in development of the insulin-responsive glucose transport system in myocytes, but is also directly involved in muscle differentiation via modulation of proNGF-p75NTR.