Myogenesis involves expression of muscle-specific transcription factors such as myogenin and myocyte enhancer factor 2 (MEF2), and is essentially regulated by fluctuations of cytosolic Ca(2+) concentration. Recently we demonstrated that molecular players of store-operated Ca(2+) entry (SOCE), stromal interacting molecule (STIM) and Orai, were fundamental in the differentiation process of post-natal human myoblasts. Besides STIM and Orai proteins, the family of transient receptor potential canonical (TRPC) channels was shown to be part of SOCE in several cellular systems. In the present study, we investigated the role of TRPC channels in the human myogenesis process. We demonstrate, using an siRNA strategy or dominant negative TRPC overexpression, that TRPC1 and TRPC4 participate in SOCE, are necessary for MEF2 expression, and allow the fusion process to generate myotubes of normal size. Conversely, the overexpression of STIM1 with TRPC4 or TRPC1 increased SOCE, accelerated myoblast fusion, and produced hypertrophic myotubes. Interestingly, in cells depleted of TRPC1 or TRPC4, the normalization of SOCE by increasing the extracellular calcium concentration or by overexpressing STIM1 or Orai1 was not sufficient to restore normal fusion process. A normal differentiation occurred only when TRPC channel was re-expressed. These findings indicate that Ca(2+) entry mediated specifically by TRPC1 and TRPC4 allow the formation of normal-sized myotubes.