MicroRNAs are a class of small noncoding RNAs that regulate gene expression post-transcriptionally either by inhibiting protein translation or by causing the degradation of target mRNAs. Current evidence indicates that miR-33b is involved in the regulation of lipid metabolism, cholesterol homeostasis, glucose metabolism and several human diseases; however, whether miR-33b contributes to the pathogenesis of human cancers and participates in the regulation of self-renewal of human cancer stem cells remains unknown. Here, we report the identification of miR-33b as a negative regulator of cell stemness and metastasis in breast cancer. Compared with paired normal breast tissues, miR-33b expression is downregulated in breast tumor samples and is inversely correlated with lymph node metastatic status. Ectopic overexpression of miR-33b in highly metastatic breast cancer cells suppresses cell self-renewal, migration and invasion in vitro and inhibits lung metastasis in vivo. Conversely, miR-33b knockdown promotes the self-renewal, migration and invasion capabilities of noncancerous mammary epithelial cells. The mechanism through which miR-33b inhibits the stemness, migration and invasion of breast cancer cells is by targeting HMGA2, SALL4 and Twist1. These data indicate that miR-33b acts as an onco-suppressive microRNA in breast cancer progression by inhibiting the stemness and metastasis of breast cancer cells.