CUDC-907 is a novel inhibitor of phosphoinositide 3-kinase and histone deacetylase. It exerts anticancer activities by inducing apoptosis and inhibiting the growth and metastases of various tumors. However, the anticancer effects of CUDC-907 on bladder cancer have not been previously reported. Thus, the present study aimed to examine the anticancer effects of CUDC-907 on 2D monolayer and 3D spheroid models of T24 cells established from highly malignant human grade III urinary bladder carcinoma and cisplatin-resistant T24R2 cells generated by 17 months of exposure to cisplatin, starting at 0.01 mu g/ml and increasing stepwise to 2 mu g/ml. CUDC-907 treatment significantly reduced the cell viabilities of the monolayer and spheroid cultures in a concentration-dependent manner. The IC50 value of CUDC-907 was higher in the bladder cancer spheroids than in the monolayers. Treatment with CUDC-907 suppressed epithelial-mesenchymal transition via decreasing vimentin and E-cadherin and consequently inhibited the migration and invasion of the bladder cancer spheroids. In addition, it promoted apoptosis and increased the expression of apoptosis-related genes, such as Bax and caspases. In conclusion, CUDC-907 exerted anticancer effects by reducing the viability, migration and invasion, and inducing apoptosis of bladder cancer spheroids. These results suggest that CUDC-907 is a potent agent for the treatment of bladder cancer.