Whole body vibration training is widely used in rehabilitation and sports activities to improve muscle strength, balance, and flexibility. However, the molecular mechanisms of vertical vibration (VV) training and their effect on the myogenesis of myoblasts remain undefined. This study was undertaken to address the hypothesis that VV can enhance the expression of ECM proteins and myogenic regulatory factors (MRFs) in myoblasts and, in turn, increase myotube formation. Using real-time PCR, Western blot analysis, and immunofluorescence studies, we examined the effect of VV treatment with frequencies of 5, 8, or 10 Hz on the expression of ECM proteins and MRFs as well as myotube formation in C2C12 myoblasts. We showed that VV stimulation is safe and effective at stimulating myogenesis in C2C12 myoblasts. The levels of expression of the ECM proteins type I collagen and decorin were the highest after VV treatment at frequencies of 8 and 10 Hz. Expression of the MRFs MyoD and myogenin increased after VV stimulation in a time- and dose-dependent manner. The total number of myotubes formed, as well as the length and the average area of myotubes, were substantially increased following VV treatment at frequencies of 8 to 10 Hz. In conclusion, VV treatment at frequencies of 8 to 10 Hz can stimulate the expression of ECM proteins and MRFs in myoblasts and, in turn, increase myotube formation.