Cancer stem cells (CSC) play a central role in cancer metastasis and development of drug resistance. miRNA are important in regulating CSC properties and are considered potential therapeutic targets. Here we report that miR-328-3p (miR-328) is significantly upregulated in ovarian CSC. High expression of miR-328 maintained CSC properties by directly targeting DNA damage binding protein 2, which has been shown previously to inhibit ovarian CSC. Reduced activity of ERK signaling in ovarian CSC, mainly due to a low level of reactive oxygen species, contributed to the enhanced expression of miR-328 and maintenance of CSC. Inhibition of miR-328 in mouse orthotopic ovarian xenografts impeded tumor growth and prevented tumor metastasis. In summary, our findings provide a novel mechanism underlying maintenance of the CSC population in ovarian cancer and suggest that targeted inhibition of miR-328 could be exploited for the eradication of CSC and aversion of tumor metastasis in ovarian cancer. SIGNIFICANCE: These findings present inhibition of miR-328 as a novel strategy for efficient elimination of CSC to prevent tumor metastasis and recurrence in patients with epithelial ovarian cancer.