Insulin-like growth factors (IGFs) can stimulate skeletal muscle differentiation. One of the molecular mechanisms underlying IGF-stimulated myogenesis is transcriptional induction of myogenin. The current work is aimed to elucidate the signaling pathways mediating the IGF effect on myogenin promoter in mouse C2C12 myogenic cells. We show that phosphatidylinositol 3-kinase (PI3K)/Akt and p70(S6K) are crucial signaling molecules mediating the stimulatory effect of IGFs on myogenin expression. We have identified three cis-elements, namely the E box, MEF2, and MEF3 sites, within the 133-base pair mouse proximal myogenin promoter that are under the control of the IGF/PI3K/Akt pathway. Simultaneous mutation of all three elements completely abolishes activation of the myogenin promoter by PI3K/Akt. We demonstrate that PI3K/Akt can increase both the MyoD and the MEF2-dependent reporter activity by enhancing the transcriptional activity of MyoD and MEF2. Interestingly, IGF1 does not enhance myogenin expression in Rhabdomyosarcoma-derived RD cells. Consistently, the constitutively active PI3K/Akt fail to activate the myogenic reporters, suggesting the IGF/PI3K/Akt pathway is defective in RD cells and the defect(s) is downstream to PI3K/Akt. This is the first time that a defect in the IGF/PI3K/Akt pathway has been revealed in RD cells which provides another clue to future therapeutic treatment of Rhabdomyosarcoma.