【Objective】 To investigate the role of microRNA-218 (miR-218) in regulating prostate cancer (PCa) cell stemness and epithelial-mesenchymal transition (EMT). 【Methods】 PCa cell line stably overexpressing miR-218 was constructed with lentivirus transfection. The expression of miR-218 was detected with real-time fluorescence quantitative polymerase chain reaction (q-PCR). The migration ability was detected with Transwell assay. The expression of EMT related proteins were detected with Western blot. The properties of cells were determined with colony formation and tumor sphere formation assays. 【Results】 The results of q-PCR showed that the mRNA level of miR-218 was significantly lower in PCa cell lines LNCaP and C4-2 than in BPH-1. Transwell assay showed that miR-218 inhibited the migration of PCa cells. Western blot showed that the expression of EMT related proteins were inhibited by miR-218. Colony formation and tumor sphere formation assays showed that overexpression of miR-218 significantly inhibited the properties of cells. 【Conclusion】 The expression of miR-218 is downregulated in PCa cell lines. miR-28 can inhibit cell migration, EMT and cancer stem cell properties.