The peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) is a highly inducible transcriptional coactivator implicated in the coordinate regulation of genes encoding enzymes involved in hepatic fatty acid oxidation, oxidative phosphorylation, and gluconeogenesis. The present study sought to assess the effects of chronic PGC-1α deficiency on metabolic flux through the hepatic gluconeogenic, fatty acid oxidation, and tricarboxylic acid cycle pathways. To this end, hepatic metabolism was assessed in wild-type (WT) and PGC-1α -/- mice using isotopomer-based NMR with complementary gene expression analyses. Hepatic glucose production was diminished in PGC-1α -/- livers coincident with reduced gluconeogenic flux from phosphoenolpyruvate. Surprisingly, the expression of PGC-1α target genes involved in gluconeogenesis was unaltered in PGC-1α -/- compared with WT mice under fed and fasted conditions. Flux through tricarboxylic acid cycle and mitochondrial fatty acid β-oxidation pathways was also diminished in PGC-1α -/- livers. The expression of multiple genes encoding tricarboxylic acid cycle and oxidative phosphorylation enzymes was significantly depressed in PGC-1α -/- mice and was activated by PGC-1α overexpression in the livers of WT mice. Collectively, these findings suggest that chronic wholeanimal PGC-1α deficiency results in defects in hepatic glucose production that are secondary to diminished fatty acid β-oxidation and tricarboxylic acid cycle flux rather than abnormalities in gluconeogenic enzyme gene expression per se. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.