Two members of the EGF receptor family, HER2 and HER3, act as key oncogenes in breast cancer cells. A MAb against HER2, trastuzumab, interferes with HER2 signaling and istherapeutically effective in humans. Here, we explored the biologic effects of an antibody against HER3 (α‐HER3ECD) in the invasive breast cancer cell lines MCF‐7ADR and MDA‐MB‐468. Pretreating the breast cancer cells with α‐HER3ECD prior to Heregulin stimulation caused significant reduction of the migratory and proliferative properties. This reduction is due to a substantial decrease in the tyrosine phosphorylation content of HER2 and to a modification of the HER2/HER3 association, which ultimately inhibits the activity of the downstream effectors phosphatidyinositol‐3‐OH‐kinase and c‐jun‐terminal kinase. Furthermore, HER3 is internalized and not activated by HRG after pretreatment with α‐HER3ECD. Our data reinforce the notion that HER3 could be a key target in cancer drug design and show the great potential of anti‐HER3 antibodies for the therapy of breast cancer and other malignancies characterized by overexpression of HER3. © 2005 Wiley‐Liss, Inc.