Bladder cancer is the most common malignancy of various heterogeneous tumor types which occur in the mucous membrane of urinary bladder. Celastrol is a bioactive natural product with anti-cancer anti-oxygenation and anti-angiogenesis functions. In this study, human bladder cancer cell line 5637 cells were treated with various concentrations of celastrol (0, 1, 2 and 4 mu M) and evaluated for cell proliferation, apoptotic rate and cell cycle distribution. CCK8 assay showed inhibited cell proliferation of 5637 cells. Besides, induced cell apoptosis and arrested cell cycle were detected by using flow cytometry. Further, Western blot analysis was conducted to explore possible mechanisms involved. Increased Bax along with decreased Bcl-2 and survivin indicated the induced apoptosis through generated ROS and disturbed MMP in 5637 cells. Nuclear translocation showed declined NF-kappa B in nucleus, indicating the inactivated NF-kappa B and inhibited Bcl-2, resulting in the apoptosis of 5637 cells. Decreased expression level of JAK2 and STAT3 indicated the blocked JAK2/STAT3 signaling pathway, corresponding to the induced cell apoptosis and arrested cell cycle. In addition, down-regulated MMP9, VEGF and VEGFR2 suggested the inhibition ability of celastrol in angiogenesis. In conclusion, we demonstrated the inhibitory ability of celastrol in cell viability of human bladder cancer cell line 5637 cells. Our study suggested a promising anti-cancer agent and provided reliable data for in vivo study.