microRNAs (miRNAs) are a class of small non-coding RNAs that can post-transcriptionally regulate gene expression and play critical roles in many important biological processes. The role of miRNAs in prostate cancer (PCa) development and pathogenesis remains largely unknown. In the present study, we showed that miR-200b was downregulated in clinical prostatic tumors when compared to normal prostate tissue and in advanced PCa cell lines when compared to normal epithelial prostatic cells. Enforced miR-200b expression suppressed PCa cell proliferation and migration and enhanced chemosensitivity to docetaxel by targeting B-cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1). Bmi-1 was detected at higher levels in PCa, and knockdown of Bmi-1 showed similar effects as miR-200b overexpression in PCa cells. Moreover, we confirmed that these effects were correlated with increased levels of E-cadherin and P16 and a reduction in vimentin expression and expression of stem cell markers (CD44 and OCT4). These findings suggest that miR-200b plays vital roles as a tumor-suppressor by targeting Bmi-1 and may be a promising therapeutic target for PCa treatment.