The nuclear factor of activated-T-cells (NFAT) family is a ubiquitous transcription factor that mediates regulation on various gene expressions. Recent studies indicate that NFAT may implicate in cancer process, mainly through its direct regulation on the cyclooxygenase-2 (COX-2) gene expression. There is also evidence suggesting another aspect of NFAT in tumor suppression. However, the according mechanism remains unknown. In this study, we used a small interfering RNA (siRNA) expression construct to study the role of NFAT3 in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation with the tumor promotion-sensitive mouse epidermal Cl41 cells. Our results showed that TPA was able to induce NFAT3 activation in Cl41 cells. Stable transfection of NFAT3 siRNA specifically reduced endogenous NFAT3 expression. At the same time, TPA-induced expression of both COX-2 and inducible nitric oxide synthase (iNOS) were blocked. However, anchorage-independent transformation in response to TPA was significantly enhanced in NFAT3 siRNA stable transfectants as compared with vector transfectants. Moreover, treatment with the iNOS specific inhibitor aminoguanidine (AG) also enhanced Cl41 cells transformation induced by TPA. As COX-2 expression is proved to be required for cell transformation in Cl41 cells in our recent studies, our results demonstrate that the inducible NFAT3-mediated iNOS upregulation represents a novel potent tumor-suppressing pathway and may contribute to the tumor suppressor functions of NFAT protein.