[Abstract] Objective: To explore the role of miR-9-5p in the biological behaviors of breast cancer cells and its possible regulatory mechanism. Methods: online OncomiR database was used to analyze the differential expression of miR-9-5p in breast cancer tissues and normal breast tissues. qPCR was used to detect the miR-9-5p expression in breast cancer cell lines and normal breast cells. Based on target gene prediction software TargetScan, ONECUT2 (one cut homeobox 2) was predicted to be the target gene of miR-9-5p. Dual luciferase reporter system was used to validate the relationship between miR-9-5p and its promising target gene ONECUT2. MDA-231 cells were transfected with miR-9-5p mimic, ONECUT2 siRNAs as well as the corresponding control sequences. The protein and mRNA levels of stemness-associated gene NOTCH1, NANOG and SOX9 (SRY (sex-determing region of Y chromosome) -Box transcription Factor 9) were detected by WB and qPCR. The effect of transfection on proliferation, apoptosis and chemo-resistance of cells was detected by BrdU method, Annexin Ⅴ method and MTS Assay, respectively. The ALDEFLUOR experiment was used to detect the effects of miR-9-5p and its target gene ONECUT2 on tumor stemness. NSG mouse breast cancer chemotherapy model was established, and the in vivo experiments further verified the effect of ONECUT2 on tumor malignant biological behaviors, such as cell stemness and chemo-resistance. Results: miR-9-5p was highly expressed in breast cancer tissues (P=0.007) and breast cancer MDA-231 cell line (P=0.0005), and was positively correlated with the poor prognosis of breast cancer patients (P=0.0016). Compared to control group, miR-9-5p could target and negatively regulate ONECUT2 expression, further increase ALDH+ cell population (P=0.0006), as well as increase the expressions of stemness-associated genes NOTCH1, NANOG and SOX9. Besides, miR-9-5p increased the anti-apoptosis ability (P=0.0003) and chemo-resistance of MDA-231 cells; however, miR-9-5p/ONECUT2 exerted no significant effect on the proliferation ability of MDA-231 cells (P>0.05). Compared with the control group, the volume of xenografts in mice of MDA-231/ONECUT2 group after DTX chemotherapy was significantly lower than that in the control group (P<0.05), and the protein expressions of NOTCH1, SOX9 and the mRNA expression of ABC transporter in the transplanted tumor tissues were significantly reduced (P<0.05 or P<0.01). Conclusions: The highly expressed miR-9-5p in breast cancer induces tumor stemness and anti-apoptotic ability by targeting ONECUT2 and enhances its resistance to chemotherapy.