The estrogen-related receptor α gene encodes a nuclear receptor protein, ERRα, whose structure is closely related to the estrogen receptors. ERRα modulates estrogen receptor (ER)-mediated signaling pathways both positively and negatively. It is selectively expressed in a variety of cell types during development and in adult tissues. We have previously shown that estrogen stimulates the expression of the ERRα gene in mouse uterus. In this study, we found that the ERRα gene is stimulated by estrogen in mouse uterus and heart but not in liver. Estrogen also stimulates the expression of ERRα in the human breast and endometrial cell lines. The human ERRα gene promoter contains multiple Sp1 binding sites, and the Sp1 protein is required for the promoter activity. The major estrogen response is mediated by a 34-bp DNA element that contains multiple steroid hormone response element half-sites (MHREs) that are conserved between the human and mouse ERRα gene promoters. Mutations made at a single or multiple sites of the MHREs abolished the ER-mediated transcription of the element in transient transfection experiments. By chromatin immunoprecipitation assay, we demonstrated the interaction between ERα and MHREs of the endogenous ERRα gene promoter in MCF-7 cells. Estrogen treatment further enhanced the association of ERα and MHREs in vivo. The present study demonstrated that the ERRα gene is a downstream target of ERα.