Estrogen-related receptors (ERR) are orphan members of the nuclear receptor superfamily most closely related to estrogen receptors (ER). Although ERalpha is a successful target for treating breast cancer, there remains an unmet medical need especially for estrogen-independent breast cancer. Although estradiol is not an ERR ligand, ER and ERR share many commonalities and overlapping signaling pathways. An endogenous ERR ligand has not been identified; however, novel synthetic ERRalpha subtype-specific antagonists have started to emerge. In particular, we recently identified a novel compound, N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2-yl)-1,3-thiazolidin-2-yl idene]-5H dibenzo[a,d][7]annulen-5-amine (termed compound A) that acts specifically as an ERRalpha antagonist. Here, we show that compound A inhibited cell proliferation in ERalpha-positive (MCF-7 and T47D) and ERalpha-negative (BT-20 and MDA-MD-231) breast cancer cell lines. Furthermore, we report the differential expression of 83 genes involved in ERRalpha signaling in MCF-7 and BT-20 breast cancer cell lines. We show that compound A slowed tumor growth in MCF-7 and BT-20 mouse xenograft models, and displayed antagonistic effects on the uterus. Furthermore, a subset of genes involved in ERRalpha signaling in vitro were evaluated and confirmed in vivo by studying uterine gene expression profiles from xenograft mice. These results suggest for the first time that inhibition of ERRalpha signaling via a subtype-specific antagonist may be an effective therapeutic strategy for ER-positive and ER-negative breast cancers.