miR-203 is an epigenetically silenced tumor-suppressive microRNA in tumors. This study was designed to investigate the effects of miR-203 on the proliferation, migration, invasion, and apoptosis of bladder cancer (BCa) cells. The expression levels of miR-203 in BCa tissues, normal adjacent tissues, and BCa cell lines were detected. BCa cells were transfected with miR-203 mimic and inhibitor to investigate the effect of miR-203 on cell functions and the epithelial-mesenchymal transition (EMT). Cotransfection with miR-203 inhibitor and shRNA of the predicted target gene Twist1 (si-Twist1) was performed to investigate the target relationship of miR-203 and Twist1. The effects of knockdown of Twist1 on cell functions were also investigated. The expression of miR-203 was significantly reduced in BCa tissues and cells, in comparison with the control. miR-203 mimic significantly reduced cell viability, invasion, migration, and EMT, and enhanced cell apoptosis. On the contrary, miR-203 inhibitor showed the opposite results. However, the administration of si-Twist1 cancelled the effect of miR-203 inhibitor on cell proliferation, apoptosis, invasion, and migration. These demonstrated that miR-203 may function as a tumor-suppressive microRNA in BCa by negatively targeting Twist1. Both Twist1 and miR-203 might be explored as potential targets for studying the mechanism related to BCa pathogenesis and therapy.