Fusion of mononuclear myoblasts to multinucleated myotubes is crucial for myogenesis. Both µ- and m-calpain are ubiquitously expressed in most cells and particularly abundant in muscle cells. Knockout of calpain-1 (catalytic subunit of µ-calpain) induced moderate platelet dysaggregation preserving the normal development and growth, though knockout of calpain-2 (m-calpain) is lethal in mice. Therefore, there should be muscle-specific function of m-calpain per se. Previous methods lack direct evidence for the involvement of m-calpain, because the specific inhibitor to m-calpain has not been developed yet and the inhibition was less potent. Here, we show that screened RNAi specifically blocked the m-calpain expression by 95% at both the protein and the activity levels. After transfecting adenovirus vector-mediated cDNA corresponding to the RNAi-induced shRNA, m-calpain in C₂C₁₂ myoblasts was knocked down with no compensatory overexpression of µ-calpain or calpain-3. The specific knockdown strongly inhibited the fusion to multinucleated myotubes. In addition, the knockdown modestly blocked ubiquitous effects, including cell-migration, spreading and alignment of central stress fiber-like structures. These results may indicate that m-calpain requiring millimolar Ca²⁺ level for the full activation plays specific roles in myogenesis, independent of µ-calpain, and leave us challenging problem in future.