Estrogen-related receptor (ERR) is a member of the nuclear receptor superfamily that has strong homology with estrogen receptor (ER) α. ERR has three subtypes (α, β, and γ) expressed in estrogen-sensitive organs, including ovary, breast, and brain. No endogenous ligands of ERRs have been identified, but these receptors share a common DNA element with ERα and control estrogen-mediated gene transcription. Recent evidence suggests a role of ERRs in estrogen-related pathophysiology, but the detailed mechanisms of ERR functions in estrogen-related tissues are unclear. Using live-cell imaging with fluorescent protein labeling, we found that only ERRβ among the ERRs exhibits a punctate intranuclear pattern overlapping with ERα following 17β-estradiol (E2)-stimulation. Fluorescence recovery after photobleaching showed significant reduction of the mobility of ligand-activated ERα with co-expression of ERRβ. Fluorescence resonance energy transfer revealed that ERRβ directly interacts with ERα. The N-terminal domain of ERRβ was identified as the region that interacts with ERα. We also found a correlation between punctate cluster formation of ERα and interaction between the receptors. Expression of ERRβ significantly repressed ERα-mediated transactivity, whereas that of other ERR subtypes had no effect on the transactivity of ERα. Consistent with this finding, E2-stimulated proliferation of MCF-7 breast carcinoma cells and bcl-2 expression was significantly inhibited by expression of ERRβ. These results provide strong evidence for a suppressive effect of ERRβ on estrogen signaling through reduction of the intranuclear mobility of ERα. The findings further suggest a unique inhibitory role for ERRβ in estrogen-dependent cellular function such as cancer cell proliferation.