The functional role of nuclear factor of activated T-cells (NFAT), while it has been extensively investigated in the immune system, remains uncertain in bladder cancer development. We here aim to assess the effects of cyclosporine A (CsA) and tacrolimus (FK506), immunosuppressants known to specifically inactivate the NFAT pathway in immune cells, on neoplastic transformation of urothelial cells. Immunohistochemistry revealed that the expression levels of NFATc1, a NFAT isoform shown to function as an oncogene in a sarcoma model, were elevated in urothelial neoplasms, compared with non-neoplastic urothelial tissues, and in low-grade and high-grade papillary urothelial carcinomas, compared with papillary urothelial neoplasms of low malignant potential. In an immortalized normal urothelial cell line SVHUC, CsA and FK506 reduced NFATc1 expression, NFAT transcriptional activity, and the expression of c-myc, a downstream target of NFATc1 signals. Treatment with CsA or FK506 in the SVHUC cells undergoing neoplastic transformation induced by exposure to a chemical carcinogen 3-methylcholanthrene resulted in strong inhibition in colony formation in vitro as well as tumor formation in NOD-SCID mice. CsA and FK506 were additionally found to up-regulate the expression of several molecules that play a protective role in bladder tumorigenesis, including p53, p21, and p27, and down-regulate that of oncogenic genes, such as cyclin D1, cyclin D3, and cyclin E, in SVHUC cells with the carcinogen challenge. Thus, CsA and FK506 likely inhibit urothelial tumorigenesis. These findings offer a potential chemopreventive approach for urothelial tumors using NFAT inhibitors. © 2015 Wiley Periodicals, Inc.