Cellular therapy using expanded autologous myoblasts is a treatment modality for a variety of diseases. In the present study, we compared the commercial skeletal muscle cell growth medium-2 (SKGM-2) with a medium designed by our group for the expansion of skeletal myoblasts. The use of an in-house medium [DMEM/F12 medium supplemented with EGF, bFGF, HGF, insulin and dexamethasone (DFEFH)] resulted in a greater number of myoblast colonies (>50%) and a 3-, 4- and 9-fold higher proliferation rate, eventually resulting in a 3-, 7- and 87-fold greater number of cells at the 1st, 2nd and 3rd passage, respectively, compared with the cells grown in SKGM-2 medium. The average CD56 expression level was higher in the myoblasts cultured in DFEFH than in those culturd in SKGM-2 medium. At the 3rd passage, lower expression levels of myostatin and considerably higher expression levels of myogenin were observed in the cells that were grown in DFEFH medium. The results of our study indicated that myoblasts cultured in both medium types displayed fusogenic potential at the 3rd passage. Furthermore, it was shown that cells cultured in DFEFH medium created myotubes with a considerably higher number of nuclei. Additionally, we observed that the fusion potential of the cells markedly decreased with the subsequent passages and that the morphology of the myoblasts differed between the 2 cultured media. Our data demonstrate that culture in the DFEFH medium leads to an approximately 90-fold greater number of myoblasts, with improved morphology and greater fusion potential, compared with culture in the commercial SKGM-2 medium