There is a global epidemic of obesity, and obesity is known to inhibit AMP-activated protein kinase (AMPK) activity and impairs myogenesis. Myogenin mediates the fusion of myoblasts into myotubes, a critical step in myogenesis. We observed that inhibition of AMPKα1 down-regulates myogenin expression and myogenesis, but the underlying mechanisms are unclear. We postulated that AMPK regulates myogenin expression through phosphorlytion of histone deacetylase 5 (HDAC5). In C2C12 cells, HDAC5 knockdown increased while HDAC5 stablization by MC1568 reduced myogenin expression. Consistently, we observed that myogenin promoter activity was negatively regulated by HDAC5. Using C2C12 cells with AMPK 1 Knockdown, and primary myoblasts prepared from wild-type and AMPKα1 KO mice, we further demonstrate that AMPK 1 regulates HDAC5 phosphorylation at Ser 259 and 498. Mutation of these two sites to Ala in HDAC5 abolished the regulatory role of AMPK 1 on myogenin expression, clearly showing the necessity of these phosphorylation sites in mediating myogenin expression. In aggregate, these data show that AMPK inhibition down-regulates myogenin transcription and myogenesis through phosphorylation of HDAC5, mediated mainly by AMPK 1. These data demonstrate that AMPK is a key molecular target for promoting myogenesis and muscular regeneration. Because drugs activating AMPK activity, such as metformin, is widely available, our finding has critical clinical implications to ensure proper muscle development and regeneration in obese subjects and under other pathophysiological conditions where AMPK activity is attenuated.