Estrogen receptors (ER alpha and ER beta) are ligand-regulated transcription factors that play critical roles in the development and progression of breast cancer by regulating target genes involved in cellular proliferation. The transcriptional activity of ER alpha and ER beta is known to be modulated by cofactor proteins. We used yeast two-hybrid system and identified NFAT3 as a novel ER beta-binding protein. NFAT3 interacted with ER alpha and ER beta both in vitro and in mammalian cells in a ligand-independent fashion. NFAT3 bound specifically to the ER beta region containing the AF-1 domain, a ligand-independent transactivation domain. Overexpression of NFAT3 enhanced both ER alpha and ER beta transcriptional activities in a ligand-independent manner, and upregulated downstream estrogen-responsive genes including pS2 and cathepsin D. Reduction of endogenous NFAT3 with NFAT3 siRNA or overexpression of NFAT3 deletion mutants which lack the ER-binding sites reduced the NFAT3 coactivation of ER alpha and ER beta. NFAT3 increased binding of ER alpha to the estrogen-responsive element (ERE) and was recruited to endogenous estrogen-responsive promoters. NFAT3 was expressed differentially in many breast cancer cell lines and overexpressed in a subset of breast cancer patients. Knockdown of endogenous NFAT3 reduced the growth of human breast cancer ZR75-1 cells in a ligand-independent manner. Taken together, these results suggest that NFAT3 may play important roles in ER signaling and represent a novel target for breast cancer therapy.