Isoflavones are excreted in human urine and can be modulated by soy-rich diets. Recently, isoflavones were suggested to have protective effects against bladder cancer cells. We sought to determine the efficacy of the antitumorigenic effects of isoflavones at concentrations found in the range of human urine excretion and compare normal urothelium and bladder cancer cells for differential cytotoxicity. A total of seven human bladder cancer cell lines and an immortalized uroepithelial cell line were used to examine the effects of genistein, daidzein, and biochanin-A, either individually or as an equal-proportion mixture regimen, on cell growth, DNA synthesis, alterations of cell cycle distribution, and induction of apoptosis. The role of cyclin B1 and cdc2 kinase in cell cycle arrest was analyzed. In addition, severe combined immunodeficient mice were used to confirm the anti-cancer effects of isoflavones in vivo. Cooperative action of isoflavones was more effective in growth inhibition and apoptosis induction than any single compound. Genistein tends to cause a dose-dependent induction of G2-M cell cycle arrest and an inhibition of cdc2 kinase activity. However, both daidzein and biochanin-A directly induced apoptosis without altering cell cycle distribution. The IC50 values in non-transformed cells were higher than those in most cancer cell lines, and the IC50 of the mixture regimen was within reach of the levels observed in urine after a soy challenge. Furthermore, both genistein and combined isoflavones exhibited a significant tumor suppressor effect in vivo (P < 0.05). The results justify the potential use of soybean foods as a practical chemoprevention approach for patients with urinary tract cancer.