In breast cancer cells, heterodimerization of HER2 and HER3 plays important and dominant roles in the functionality and transformation of HE R-mediated pathways, in particular the PI3K/Akt survival pathway. HER3 was considered as a major signaling hub in HER2-amplified cancers. Inhibition of HER3 expression may therefore represent a rational therapeutic approach to breast cancers where HER2/HER3-mediated signaling plays a role in tumorigenesis and progression. miRNAs exerts important roles in regulating gene expressions by binding to and repressing target mRNAs. Here we reported that miRNA-450b-3p inhibits HER3 expression by directly targeting 3' UTR of HER3 mRNA and represses the downstream signal transductions of HER family. Overexpression of miRNA-450b-3p in SKBR3 cells inhibits cells clonogenic potential and enhances their sensitivity to trastuzumab, a monoclonal antibody that binds to the HER2 receptor, or doxorubicin through repressing proliferative signal pathways mediated by HER3/HER2/PI3K/AKT. Furthermore, we found that breast cancer patients with tumors that demonstrating upregulated HER3 (>2-fold) and downregulated miR-450b-3p (>2-fold) expressions compared with the paired adjacent non-tumorous tissues showed significantly poorer overall survival (P < 0.05). Our study identified miRNA-450b-3p as a new tumor repressor and also provided some evidences suggesting that downregulation of miR-450b-3p expression with concurrent overexpression of HER3 may serve as a prognostic biomarker for poor overall survival in breast cancer patients.