Abnormal activation of human epidermal growth factor receptor 2 (HER2; ErbB-2) in breast tumors results in increased metastasis and angiogenesis, as well as reduced survival. Here, we show that angiopoietin-2 (Ang-2) expression correlates with HER2 activity in human breast cancer cell lines. Inhibiting HER2 activity with anti-HER2 monoclonal antibody trastuzumab (Herceptin) or HER2 short interfering RNA in tumor cells down-regulates Ang-2 expression. Consistent with the important roles of AKT and mitogen-activated protein kinase in the HER2 signaling pathway, AKT and ERK mitogen-activated protein kinase (MAPK) kinase activity is necessary for Ang-2 up-regulation by HER2. Moreover, overexpression of HER2 protein up-regulates Ang-2 expression. Heregulin-beta1-induced Ang-2 up-regulation is abrogated when AKT and ERK kinase activity are blocked. Immunohistochemical analysis of HER2 and Ang-2 proteins in human breast carcinomas shows that Ang-2 expression in breast cancer correlates with HER2 expression. These studies provide evidence that the Ang-2 gene is regulated by HER2 activity in breast cancer, and propose an additional mechanism for HER2 contributing to tumor angiogenesis and metastasis.