Increasing evidence indicates that microRNAs (miRNAs) have essential roles in various biological processes, including proliferation, migration, invasion, cell cycle progression and apoptosis. It is considered that miRNA de-regulation contributes to tumor progression and metastasis in various cancers, and MiR-203a has been identified as a tumor suppressor in cancers, such as glioma, gastric cancer and hepatocellular carcinoma. Herein, we established that miR-203a expression is significantly lower in bladder cancer tissues than in adjacent normal tissues, and that low miR-203a expression is associated with poor patient outcome. The over-expression of miR-203a inhibited bladder cancer cell proliferation, invasion, migration and EMT in vitro, and its up-regulation led to bladder cancer cell cycle arrest and apoptosis. This over-expression also inhibited the PI3K/Akt signaling pathway. Bioinformatics prediction software and luciferase reporter assay then confirmed that SIX4 is a direct target of miR-203a. We established negative correlation between SIX4 expression and miR-203a expression in bladder cancer tissues, and SIX4 silencing caused effects similar to miR-203a up-regulation Furthermore, SIX4 over-expression diminished the effects of miR-203a on bladder cancer cells in vitro. In summary, our study determined that miR-203a down-regulation is closely related to tumorigenesis in bladder cancer; thus suggesting that miR-203a is a potential prognostic marker and a potential target in bladder cancer treatment.