MicroRNAs play key roles during various crucial cell processes, such as proliferation, migration, and invasion. In addition, microRNAs have been shown to possess oncogenic and tumor suppressive functions in human cancers. Increasing evidence has clarified that miR-149-3p, a novel cancer-related microRNA, plays an important role in suppression of proliferation, migration, and invasion; however, the effect and mechanisms underlying the miR-149-3p effect in bladder cancer (BCa) remain unclear. In the current study we found that the increased expression of miR-149-3p significantly suppressed cell proliferation, migration, and invasion ability in BCa. The suppressive effect was related to S100A4. A further investigation showed that miR-149-3p negatively regulated S100A4, as verified by the luciferase reporter assay. Furthermore, our study showed that S100A4 mediated the anti-metastatic effects of miR-149-3p on proliferation, migration, and invasion of BCa cells. Analysis of a xenograft mouse model showed that miR-149-3p expression significantly decreased tumor growth by targeting S100A4. Taken together, these data indicate that S100A4 promotes cell growth, migration, and invasion and can by reversed by miR-149-3p in BCa.