The orphan nuclear receptor, estrogen-related receptor beta (ERRbeta), shares a high degree of amino acid identity with estrogen receptor alpha (ERalpha). Although ERRbeta has been shown to be critical in embryo development, little is known about its functions and target genes. Here we report that the newly identified and most common human ortholog of ERRbeta--short-form hERRbeta (SFhERRbeta) potently represses the transcriptional activity of NF-E2 Related Factor 2 (Nrf2) on antioxidant response element (ARE)-mediated gene expression. Nrf2 is a main regulator of the expression of phase II detoxifying enzymes and antioxidant proteins in the cellular protection against oxidative stress. SFhERRbeta is the most potent inhibitor of Nrf2 transcriptional activity among the three ERR family members, ERRalpha, ERRbeta and ERRgamma. Additional analyses revealed that SFhERRbeta repressed Nrf2 activity likely through physical interaction in a complex with Nrf2, not by competing for the ARE DNA-binding sites, nor by decreasing Nrf2 protein concentration. By confocal immunofluorescence microscopy, SFhERRbeta alters the subcellular localization of Nrf2. Analyses using SFhERRbeta deletion mutants showed that SFhERRbeta interacts with Nrf2 through multiple sites. Our findings suggest that ERRbeta plays a novel functional role in the Nrf2-ARE pathway. By acting as a repressor of Nrf2, ERRbeta may be useful as a therapeutic target in cancer chemoprevention studies.