Estrogen-related receptor (ERR), a member of nuclear receptor superfamily, has high homology with estrogen receptor (ER) α and consists of three subtypes (α, β, and γ). Our previous research confirmed the expression of ERRs in estrogen-sensitive brain regions including preoptic area and hypothalamus. While no endogenous ligands of ERRs are identified to date, they share the common DNA element with ERα and control ERα-mediated gene transcription. Recent evidences suggest the impact of ERRs on estrogen-related pathophysiology, however, the detailed mechanism of ERR functions in estrogen-related tissues has not been fully explained. Using living-cell imaging, we showed that among ERRs, only ERRβ exhibits punctate intranuclear pattern that overlaps with ERα following estradiol (E2) stimulation. Fluorescence recovery after photobleaching analyses demonstrated the significant reduction of the mobility of E2-activated ERα by co-expression of ERRβ. Deletion mutant experiments clearly identified the N-terminal domain of ERRβ is responsible to interact with ERα. We also revealed the correlation between the cluster formation of ERα and the interaction of the two receptors. The expression of ERRβ significantly repressed ERα-mediated transcriptional activity while other subtypes of ERRs exhibited no effect on the transactivity of ERα. Consistently, E2-stimulated proliferation of MCF-7 cells was significantly inhibited by the expression of ERRβ. These results provide strong evidence for suppressive effect of ERRβ on estrogen signaling through the reduction of intranuclear mobility of ERα. Present findings further suggest the unique inhibitory role for ERRβ in estrogen-dependent cellular function.