SOX2 (Sry-related high-mobility box SOX-2) is a transcription factor, which is essential for maintaining the cancer cell stemness. However, the role of microRNAs targeting SOX2 in cancer cell stemness remains unclear. We examined the effect of miR-590-5p, which targeted SOX2, on the breast cancer cell stemness and metastasis.We predicted and screened microRNA targeting SOX2, and further investigated the regulatory role of miR-590-5p on the level of SOX2 with Western blot, luciferase reporting assay and qRT-PCR analysis. Flow cytometry was performed to detect the effect of miR-590-5p on the breast cancer stem cell population with ALDEFLUOR Assay. We inoculated the breast cancer cells transfected with or without miR-590-5p to NOD/SCID mice to detect the tumorigenicity in vivo. Finally, forty-nine pairs of breast cancer samples and adjacent noncancerous tissues were obtained, and immunohistochemistry (IHC) with SOX2 antibody and qRT-PCR assay were used to quantify the expression of miR-590-5p in breast cancer samples.miR-590-5p significantly downregulated the SOX2 protein expression, and inhibition of miR-590-5p increased SOX2 expression. The luciferase reporter assay indicated that miR-590-5p decreased the SOX2 3'UTR (3' untranslated region) reporter activity but not the luciferase activity of the mutant reporter, in which the binding sites for miR-590-5p were mutated. ALDEFLUOR Assay showed that miR-590-5p significantly decreased breast cancer stem cells population. NOD/SCID nude mice experiments indicated that miR-590-5p significantly inhibited tumorigenicity of breast cancer cells. IHC assay and qRT-PCR suggested that miR-590-5p expression was downregulated in breast cancer patients, and negatively correlated with SOX2.miR-590-5p inhibited breast cancer cell stemness through targeting SOX2. Our study indicated that miR-590-5p might be a useful strategy for breast cancer treatment.