Blockade of the human epidermal growth factor receptor 3 (HER3) and of the downstream phosphatidylinositide 3-kinase (PI3K)/AKT pathway is a prerequisite for overcoming drug resistance and to develop novel treatments for cancers that are not eligible for the currently approved targeted therapies. To this end, we generated specific antibodies (Abs) against domain 1 (D1) and domain 3 (D3) of HER3 that recognize epitopes that do not overlap with the neuregulin-binding site. The fully human H4B-121 Ab and the mouse monoclonal Abs 16D3-C1 and 9F7-F11 inhibited tumor growth in nude mice xenografted with epidermoid, pancreatic, or triple-negative breast cancer cells. The combination of one anti-HER3 Ab and trastuzumab improved tumor growth inhibition in mice xenografted with HER2low cancer cell lines, for which trastuzumab alone shows no or moderate efficiency. Ab-induced disruption of tumor growth was associated with G1 cell cycle arrest, proliferation inhibition, and apoptosis of cancer cells. Anti-HER3 Abs blocked HER2/HER3 heterodimerization and HER3 phosphorylation at the cell membrane, leading to inhibition of phosphorylation of the downstream AKT targets murine double minute 2, X-linked inhibitor of apoptosis, and forkhead box O1. This study demonstrates that anti-HER3 D1 and D3 Abs could represent a new option for immunotherapy of pancreatic and triple-negative breast cancers.