The estrogen-related receptor alpha (ERRalpha) is an orphan member of the superfamily of nuclear hormone receptors expressed in tissues that preferentially metabolize fatty acids. Despite the molecular characterization of ERRalpha and identification of target genes, determination of its physiological function has been hampered by the lack of a natural ligand. To further understand the in vivo function of ERRalpha, we generated and analyzed Estrra-null (ERRalpha-/-) mutant mice. Here we show that ERRalpha-/- mice are viable, fertile and display no gross anatomical alterations, with the exception of reduced body weight and peripheral fat deposits. No significant changes in food consumption and energy expenditure or serum biochemistry parameters were observed in the mutant animals. However, the mutant animals are resistant to a high-fat diet-induced obesity. Importantly, DNA microarray analysis of gene expression in adipose tissue demonstrates altered regulation of several enzymes involved in lipid, eicosanoid, and steroid synthesis, suggesting that the loss of ERRalpha might interfere with other nuclear receptor signaling pathways. In addition, the microarray study shows alteration in the expression of genes regulating adipogenesis as well as energy metabolism. In agreement with these findings, metabolic studies showed reduced lipogenesis in adipose tissues. This study suggests that ERRalpha functions as a metabolic regulator and that the ERRalpha-/- mice provide a novel model for the investigation of metabolic regulation by nuclear receptors.