It has previously been known that transcription of the PGC-1α gene can be either inhibited or stimulated by p38 MAP kinase (p38 MAPK). To determine whether p38 MAPK plays an inhibitory or stimulatory role in PGC-1α gene transcription, we further investigated the role of p38 MAPK in this study. Our results showed that the basal level of p38 MAPK phosphorylation was increased in gastrocnemius of mice under HFD and that p38 MAPK stimulated PGC-1α gene transcription in C₂C₁₂ myotubes. Our results also provided new mechanisms in myotubes that the p38 MAPK-induced PGC-1α gene transcription was mediated by CREB. In exploring the role of the Akt-dependent insulin signaling on PGC-1α gene transcription, we found that the basal Akt-dependent signaling was increased in gastrocnemius of mice under HFD. The p38 MAPK-induced PGC-1α gene transcription was prevented by insulin. Insulin suppression of PGC-1α gene transcription was neutralized by overexpression of the constitutively nuclear form of FoxO1. Finally, we located three insulin response elements (IREs) in the PGC-1α promoter, and mutations of these IREs abolish or blunt activity of the PGC-1α promoter. Together, our results show that transcription of the PGC-1α gene is balanced by different intracellular signaling pathways.