Bladder cancer is one of the most commonly diagnosed urological malignancies. Acquired resistance to chemotherapy is a great barrier for achieving successful treatment of bladder cancer. In the present study, we investigated the effect and mechanisms of icaritin, a flavonol glycoside derived from genus Epimedium, against human bladder cancer cells. It was found that despite the low cytotoxicity in normal human HEK293 cells, icaritin significantly inhibited the proliferation and colony formation of BT5637 and T24 bladder cancer cells time- and dose-dependently compared to the DMSO vehicle control. Moreover, cell viability monitored through mitochondrial membrane potential was inhibited markedly after icaritin treatment. Further investigation indicated that icaritin may inhibit epirubicin (EPI)-induced autophagy, and acted synergistically with EPI to suppress the proliferation of BT5637 and T24 cells. These findings suggest that icaritin may prove to be a novel potent therapeutic agent for the treatment of bladder cancer.