The regulation of estrogen-related receptor (ERR) transcriptional activity is poorly understood. To explore the underlying mechanism, we sought to isolate ERRα-binding protein(s). In a yeast two-hybrid screen, we identified a novel protein that has been characterized as a retinoic acid resistance factor (RaRF) (manuscript in-preparation). A specific interaction between RaRF and ERRα was confirmed in a GST pull-down assay in vitro and immunoprecipitation (IP) in mammalian cells. Further yeast two-hybrid assays and IP analyses indicated that the C-terminus of ERRα is required for RaRF binding. Consistent with our interaction data, transfection of RaRF significantly reduced the ability of ERRα, but not ERRγ, to transactivate an ERR-responsive luciferase reporter. In contrast, down-regulation of RaRF using shRNA increased ERRα activity without affecting that of ERRγ. RaRF was subsequently shown to repress the expression of the ERR target gene pS2. Further fluorescence microscopy revealed that ERRα or ERRγ is normally expressed in the nucleoplasm, with ERRα, but not ERRγ, translocating to the nucleolus when RaRF is expressed. Taken together, our data suggest that RaRF sequesters ERRα in the nucleolus through a specific interaction, thereby inhibiting its transcriptional activity.