Abstract The objectives were to investigate the roles of different calpains and protein kinase C (PKC) isoforms in muscle differentiation. Concentrations of μ- and m-calpain increased significantly whereas PKCα and δ declined significantly during L8 myoblast differentiation. Both μ-calpain and m-calpain antisense oligonucleotides inhibited myotube formation and creatine kinase activity during L8 myoblast differentiation. These results implied that both μ- and m-calpain were involved in L8 myoblast differentiation. To investigate the involvement of calpain in regulation of PKC concentrations, μ-calpain antisense oligonucleotides were added to L8 myoblasts. PKCα remained unchanged and PKCδ declined. By adding m-calpain antisense oligonucleotides instead, PKCα level remained unchanged and PKCδ concentrations increased significantly during differentiation. These results suggest that PKCα, but not PKCδ, is the substrate for μ-calpain and PKCα and δ are the substrates for the m-calpain. In addition, more phosphorylated myogenin was found in day 2 antisense oligonucleotides treated L8 cells. It is concluded that the decline of PKCα mediated by m- and μ-calpain is essential for L8 myoblast differentiation. The decline of PKC during myoblast differentiation may cause hypo-phosphorylation of myogenin, which in turn activates muscle-specific genes during myogenesis.