Mulberry fruit water extract (MWE) has been reported to synergistically enhance the cytotoxic effect of paclitaxel by promoting mitotic catastrophe to induce apoptosis in bladder cancer cells in our previous work. The aim of this study was to evaluate and to mechanistically explore the effects of MWE on bladder cancer responses to ionizing radiation (IR) by treating TSGH 8301 bladder carcinoma cells with MWE after exposing to IR. The results of MTT assay showed a synergistic cytotoxicity of IR with the co-treatment of MWE (IR/MWE) by inducing G2/M phase arrest as demonstrated by flow cytometry analysis in TSGH 8301, HT1367 and HT1197 bladder carcinoma cells lines. The IR/MWE-treated cells expressed increased levels of the G2/M phase arrest-related proteins cdc2/cyclin B1 and displayed giant multinucleated morphology, a typical characteristic of mitotic catastrophe. Immunofluorescent confocal microscopy revealed that the combined strategy inhibited Aurora B phosphorylation through Ras/Raf/MEK/ERK signaling cascade as demonstrated by Western blotting analysis. IR/MWE also caused an inhibitory effect on Plk1 and the subsequent downstream regulator RhoA repression and Cep55 induction, which would influence cell cycle progression in the early steps of cytokinesis. A profound tumor growth suppression and inactivation of Aurora B activity in the tumor tissues by IR/MWE treatment were confirmed in the TSGH 8301 xenograft model in vivo. These data demonstrated that MWE could be an effective auxiliary to synergize with radiation on the anticancer efficacy by promoting mitotic catastrophe through inhibition of Aurora B, providing a novel and effective therapeutic option for bladder cancer management.