Insulin-like growth factor (IGF-I) activates signaling pathways that increase the expression of muscle-specific genes in differentiating myoblasts. Induction of skeletal -actin expression occurs during differentiation through unknown mechanisms. The purpose of this investigation was to examine the mechanisms that IGF-I uses to induce skeletal -actin gene expression in C2C12 myoblasts. IGF-I increased skeletal -actin promoter activity by 107% compared with the control condition. Ni⁺ (T-type Voltage Gated Ca²⁺ Channel (VGCC) inhibitor) reduced basal-induced activation of the skeletal -actin promoter by 84% and nifedipine (L-type VGCC inhibitor) inhibited IGF-I-induced activation of the skeletal -actin promoter by 29-48%. IGF-I failed to increase skeletal -actin promoter activity in differentiating dysgenic (lack functional L-type VGCC) myoblasts. 30 mM K⁺ and 30 mM K⁺+IGF-I increased skeletal -actin promoter activity by 162% and 76% compared with non-IGF-I or IGF-I only conditions, respectively. IGF-I increased calcineurin activity, which was inhibited by cyclosporine A (CsA). Further, CsA inhibited K⁺+IGF-I-induced activation of the skeletal -actin promoter. Constitutively active calcineurin increased skeletal -actin promoter activity by 154% and rescued the nifedipine-induced inhibition of L-type VGCC, but failed to rescue the Ni⁺-inhibition of T-type VGCC. IGF-I-induced Nuclear Factor of Activated T-cells (NFAT) transcriptional activity was not inhibited by nifedipine or Ni⁺. IGF-I failed to increase serum response factor (SRF) transcriptional activity, however SRF activity was reduced in the presence of Ni⁺. These data suggest that IGF-I-induced activation of the skeletal -actin promoter is regulated by the L-type VGCC and calcineurin, but independent of NFAT transcriptional activity as C2C12 myoblasts differentiate into myotubes.