HER2 (ErbB2) has been reported to be overexpressed in 20–30% of breast cancer and confers poor survival because of high proliferation and metastasis rates. MicroRNAs are small noncoding RNAs that are responsible for the post-transcriptional regulation of target genes. We found miR-199b-5p inhibited HER2 expression by direct targeting its 3′-untranslated region (3′UTR) in breast cancer cells. In addition, miR-199b-5p inhibited HER2 downstream signaling by ERK1/2 and AKT pathways in breast cancer cells. Besides, transwell migration, wound healing, and clonogenicity were obviously inhibited by overexpression of miR-199b-5p in HER2-positive breast cancer cells. We also found that miR-199b-5p could enhance the suppression of trastuzumab on cell migration and clonogenicity. These results suggest that miR-199b-5p may have the potential to be a novel important alternative therapeutic target for HER2-positive breast cancer. J. Cell. Biochem. 114: 1457–1463, 2013. © 2013 Wiley Periodicals, Inc.