Previous investigations show that intracerebroventricular administration of a potent inhibitor of fatty acid synthase, C75, increases the level of its substrate, malonyl-CoA, in the hypothalamus. The “malonyl-CoA signal” is rapidly transmitted to skeletal muscle by the sympathetic nervous system, increasing fatty acid oxidation, uncoupling protein-3 (UCP3) expression, and thus, energy expenditure. Here, we show that intracerebroventricular or intraperitoneal administration of C75 increases the number of mitochondria in white and red (soleus) skeletal muscle. Consistent with signal transmission from the hypothalamus by the sympathetic nervous system, centrally administered C75 rapidly (≤2 h) up-regulated the expression (in skeletal muscle) of the β-adrenergic signaling molecules, i.e., norepinephrine, β₃-adrenergic receptor, and cAMP; the transcriptional regulators peroxisomal proliferator activator regulator γ coactivator 1α (PGC-1α) and estrogen receptor-related receptor α (ERRα); and the expression of key oxidative mitochondrial enzymes, including pyruvate dehydrogenase kinase, medium-chain length fatty acyl-CoA dehydrogenase, ubiquinone–cytochrome c reductase, cytochrome oxidase, as well as ATP synthase and UCP3. The role of PGC-1α in mediating these responses in muscle was assessed with C2C12 myocytes in cell culture. Consistent with the in vivo response, adenovirus-directed expression of PGC-1α in C2C12 muscle cells provoked the phosphorylation/inactivation and reduced expression of acetyl-CoA carboxylase 2, causing a reduction of the malonyl-CoA concentration. These effects, coupled with an increased carnitine palmitoyltransferase 1b, led to increased fatty acid oxidation. PGC-1α also increased the expression of ERRα, PPARα, and enzymes that support mitochondrial fatty acid oxidation, ATP synthesis, and thermogenesis, apparently mediated by an increased expression of UCP3.