Hormonal and nutrient regulation of hepatic gluconeogenesis mainly occurs through modulation of the transcriptional coactivator PGC-1α. The identity of endogenous proteins and their enzymatic activities that regulate the functions and form part of PGC-1α complex are unknown. Here, we show that PGC-1α is in a multiprotein complex containing the acetyltransferase GCN5. PGC-1α is directly acetylated by GCN5 resulting in a transcriptionally inactive protein that relocalizes from promoter regions to nuclear foci. Adenoviral-mediated expression of GCN5 in cultured hepatocytes and in mouse liver largely represses activation of gluconeogenic enzymes and decreases hepatic glucose production. Thus, we have identified the endogenous PGC-1α protein complex and provided the molecular mechanism by which PGC-1α acetylation by GCN5 turns off the transcriptional and biological function of this metabolic coactivator. GCN5 might be a pharmacological target to regulate the activity of PGC-1α, providing a potential treatment for metabolic disorders in which hepatic glucose output is dysregulated.