The functional role of nuclear factor of activated T-cells (NFAT), a key regulator of the immune response, in bladder cancer progression remains uncertain. In this study, we assessed biological significance of NFAT in human bladder cancer. Immunohistochemistry detected nuclear/cytoplasmic NFATc1 signals in 14 (21.5%)/34 (52.3%), respectively, of 65 muscle-invasive bladder carcinomas and showed that patients with nuclear NFATc1-positive tumor had a significantly higher risk of disease progression (P = 0.006). In bladder cancer cell lines, cyclosporine A (CsA) and tacrolimus (FK506), immunosuppressant drugs/non-selective NFAT inhibitors, attenuated NFATc1 expression and its nuclear translocation, NFAT transcriptional activity, and the expression of cyclooxygenase-2 and c-myc, downstream targets of NFATc1. NFAT inhibition via NFATc1-small interfering RNA (siRNA) or treatment with these NFAT inhibitors resulted in decreases in cell viability/colony formation, cell migration/invasion, and the expression/activity of MMP-2 and MMP-9, as well as an increase in apoptosis, in the parental/control lines. No significant additional inhibition in the viability and invasion of NFATc1-siRNA cells was seen. In xenograft-bearing mice, CsA and FK506 significantly retarded tumor growth. These results suggest that NFATc1 plays an important role in bladder cancer outgrowth. Thus, NFATc1 inactivation, especially using CsA and FK506, has the potential of being a therapeutic approach for bladder cancer.