NFAT3 belongs to the NFAT family of transcription factors playing important roles in the development of several organ systems and was found to act as a transcriptional coactivator of estrogen receptors (ERalpha and ERbeta) in breast cancer cells. Since some cofactors of transcription factors show cell or tissue type-specific effects on transcriptional regulation, we investigated the effect of NFAT3 on the transcriptional activity of ERs in different cell lines originated from kidney. Surprisingly, overexpression of NFAT3 in these cell types decreased dose-dependently both ERalpha and ERbeta transcriptional activities in a ligand-independent manner. Knockdown of endogenous NFAT3 using NFAT3 small interfering RNA (siRNA) increased ER transcriptional activities. NFAT3 deletion mutants lacking the ER-binding sites completely abolished the NFAT3 repression of ERalpha and ERbeta transcriptional activities. Replacement of Ser168 and Ser170, the amino acid residues on which NFAT3 can be phosphorylated, with Ala did not change the ability of NFAT3 to inhibit the transcriptional activity of ERalpha and ERbeta. Taken together, these results demonstrate that NFAT3 is a new kind of cofactor that displays dual transcription modulation mode dependent on tissue types.