The miR-133b, a commonly recognized muscle-specific miRNA, was reported to be deregulated in many kinds of cancers. However, its potential roles in tumorigenesis remain greatly elusive. Herein, we demonstrate that miR-133b is significantly suppressed in human breast cancer specimens, which is reversely correlated to histological grade of the cancer. Ectopic expression of miR-133b suppresses clonogenic ability and metastasis-relevant traits in vitro, as well as carcinogenesis and pulmonary metastasis in vivo. Further studies have identified Sox9, c-MET, and WAVE2 as direct targets of miR-133b, in which Sox9 contributes to all miR-133b-endowed effects including cell proliferation, colony formation, as well as cell migration and invasion in vitro. Moreover, re-expression of Sox9 reverses miR-133b-mediated metastasis suppression in vivo. Taken together, these findings highlight an important role for miR-133b in the regulation of tumorigenesis and metastatic potential of breast cancer and suggest a potential application of miR-133b in cancer treatment.