The transcriptional coactivator PGC-1α is a key regulator of energy metabolism, yet little is known about its role in control of substrate selection. We found that physiological stimuli known to induce PGC-1α expression in skeletal muscle coordinately upregulate the expression of pyruvate dehydrogenase kinase 4 (PDK4), a negative regulator of glucose oxidation. Forced expression of PGC-1α in C2C12 myotubes induced PDK4 mRNA and protein expression. PGC-1α-mediated activation of PDK4 expression was shown to occur at the transcriptional level and was mapped to a putative nuclear receptor binding site. Gel shift assays demonstrated that the PGC-1α-responsive element bound the estrogen-related receptor α (ERRα), a recently identified component of the PGC-1α signaling pathway. In addition, PGC-1α was shown to activate ERRα expression. Chromatin immunoprecipitation assays confirmed that PGC-1α and ERRα occupied the mPDK4 promoter in C2C12 myotubes. Additionally, transfection studies using ERRα-null primary fibroblasts demonstrated that ERRα is required for PGC-1α-mediated activation of the mPDK4 promoter. As predicted by the effects of PGC-1α on PDK4 gene transcription, overexpression of PGC-1α in C2C12 myotubes decreased glucose oxidation rates. These results identify the PDK4 gene as a new PGC-1α/ERRα target and suggest a mechanism whereby PGC-1α exerts reciprocal inhibitory influences on glucose catabolism while increasing alternate mitochondrial oxidative pathways in skeletal muscle. Copyright © 2005, American Society for Microbiology. All Rights Reserved.