In this study, we have investigated the effects of insulin-like growth factor-I (IGF-I) on cellular responses of primary human skeletal muscle cells and mouse C2C12 myoblasts. In human muscle, IGF-I stimulated proliferation and fusion of the cells and the expression of the differentiation marker desmin. These effects were completely inhibited by Rottlerin, the inhibitor of the protein kinase C (PKC)δ, but were not affected by the inhibition of the mitogen-activated protein kinase (MAPK) or the phosphatidylinositide 3-kinase (PI-3K) pathways. Furthermore, IGF-I initiated the selective translocation of PKCδ to the nucleus. In C2C12 myoblasts, the growth-promoting effects of IGF-I were abrogated by inhibition of PKCδ, but not by the inhibition of the PI-3K system. However, in contrast to the human data, the MAPK inhibitor PD098059 partially (yet significantly) also inhibited the action of IGF-I and, furthermore, IGF-I induced phosphorylation of the MAPK Erk-1/2. In addition, overexpression of constitutively active form of PKCδ in C2C12 cells fully mimicked, whereas overexpression of kinase inactive mutant of the isoform prevented the action of IGF-I. Finally, the inhibition of PKCδ suspended the IGF-I-induced phosphorylation of Erk-1/2 and, moreover, the inhibition of the MAPK pathway partially (yet significantly) inhibited the accelerated growth of C2C12 cells overexpressing PKCδ. Taken together, these results demonstrate a novel, central and exclusive involvement of PKCδ in mediating the action of IGF-I on human skeletal muscle cells, with an additional yet PKCδ-dependent contribution of the MAPK pathway on C2C12 myoblasts.