The transcriptional coactivator PGC-1alpha 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-1alpha expression in skeletal muscle coordinately upregulate the expression of pyruvate dehydrogenase kinase 4 (PDK4), a negative regulator of glucose oxidation. Forced expression of PGC-1alpha in C(2)C(12) myotubes induced PDK4 mRNA and protein expression. PGC-1alpha-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-1alpha-responsive element bound the estrogen-related receptor alpha (ERRalpha), a recently identified component of the PGC-1alpha signaling pathway. In addition, PGC-1alpha was shown to activate ERRalpha expression. Chromatin immunoprecipitation assays confirmed that PGC-1alpha and ERRalpha occupied the mPDK4 promoter in C(2)C(12) myotubes. Additionally, transfection studies using ERRalpha-null primary fibroblasts demonstrated that ERRalpha is required for PGC-1alpha-mediated activation of the mPDK4 promoter. As predicted by the effects of PGC-1alpha on PDK4 gene transcription, overexpression of PGC-1alpha in C(2)C(12) myotubes decreased glucose oxidation rates. These results identify the PDK4 gene as a new PGC-1alpha/ERRalpha target and suggest a mechanism whereby PGC-1alpha exerts reciprocal inhibitory influences on glucose catabolism while increasing alternate mitochondrial oxidative pathways in skeletal muscle.