It has been reported that feeding mice resveratrol activates AMPK and SIRT1 in skeletal muscle leading to deacetylation and activation of PGC-1α, increased mitochondrial biogenesis, and improved running endurance. This study was done to further evaluate the effects of resveratrol, SIRT1, and PGC-1α deacetylation on mitochondrial biogenesis in muscle. Feeding rats or mice a diet containing 4 g resveratrol/kg diet had no effect on mitochondrial protein levels in muscle. High concentrations of resveratrol lowered ATP concentration and activated AMPK in C₂C₁₂ myotubes, resulting in an increase in mitochondrial proteins. Knockdown of SIRT1, or suppression of SIRT1 activity with a dominant-negative (DN) SIRT1 construct, increased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C₂C₁₂ cells. Expression of a DN SIRT1 in rat triceps muscle also induced an increase in mitochondrial proteins. Overexpression of SIRT1 decreased PGC-1α acetylation, PGC-1α coactivator activity, and mitochondrial proteins in C₂C₁₂ myotubes. Overexpression of SIRT1 also resulted in a decrease in mitochondrial proteins in rat triceps muscle. We conclude that, contrary to some previous reports, the mechanism by which SIRT1 regulates mitochondrial biogenesis is by inhibiting PGC-1α coactivator activity, resulting in a decrease in mitochondria. We also conclude that feeding rodents resveratrol has no effect on mitochondrial biogenesis in muscle.