We investigated the role of NF-kB in the development and progression of urothelial cancer as well as cross-talk between NF-kB and androgen receptor (AR) signals in urothelial cells. Immunohistochemistry in surgical specimens showed that the expression levels of NF-kB/p65 (P ¼ 0.015)/phospho-NF-kB/ p65 (P < 0.001) were significantly elevated in bladder tumors, compared with those in nonneoplastic urothelial tissues. The rates of phospho-NF-kB/p65 positivity were also significantly higher in high-grade (P ¼ 0.015)/muscle-invasive (P ¼ 0.033) tumors than in lower grade/non–muscle-invasive tumors. Additionally, patients with phospho-NF-kB/p65-positive muscle-invasive bladder cancer had significantly higher risks of disease progression (P < 0.001) and cancer-specific mortality (P ¼ 0.002). In immortalized human normal urothelial SVHUC cells stably expressing AR, NF-kB activators and inhibitors accelerated and prevented, respectively, their neoplastic transformation induced by a chemical carcinogen 3-methyl-cholanthrene. Bladder tumors were identified in 56% (mock), 89% (betulinic acid), and 22% (parthenolide) of N-butyl-N-(4-hydroxybutyl)nitrosamine-treated male C57BL/6 mice at 22 weeks of age. NF-kB activators and inhibitors also significantly induced and reduced, respectively, cell proliferation/migration/ invasion of AR-positive bladder cancer lines, but not AR-knockdown or AR-negative lines, and their growth in xenograft-bearing mice. In both nonneoplastic and neoplastic urothelial cells, NF-kB activators/inhibitors upregulated/downregulated, respectively, AR expression, whereas AR overexpression was associated with increases in the expression levels of NF-kB/ p65 and phospho-NF-kB/p65. Thus, NF-kB appeared to be activated in bladder cancer, which was associated with tumor progression. NF-kB activators/inhibitors were also found to modulate tumorigenesis and tumor outgrowth in AR-activated urothelial cells. Accordingly, NF-kB inhibition, together with AR inactivation, has the potential of being an effective chemopreventive and/or therapeutic approach for urothelial carcinoma.