HER3 belongs to the epidermal growth factor receptor (EGFR) family and is known to form an active heterodimer with other three family members EGFR, HER2, and HER4. HER3 is overexpressed in lung, breast, colon, prostate, and gastric cancers. In the present study, we developed and validated an anti‑HER3 monoclonal antibody (mAb), H3Mab‑17 (IgG2a, kappa), by immunizing mice with HER3‑overexpressed CHO‑K1 cells (CHO/HER3). H3Mab‑17 was found to react specifically with endogenous HER3 in colorectal carcinoma cell lines, using flow cytometry. The KD for H3Mab‑17 in CHO/HER3 and Caco‑2 (a colon cancer cell line) were determined to be 3.0x10‑9 M and 1.5x10‑9 M via flow cytometry, respectively, suggesting high binding affinity of H3Mab‑17 to HER3. Then, we assessed the H3Mab‑17 antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC) against Caco‑2, and evaluated its antitumor capacity in a Caco‑2 xenograft model. In vitro experiments revealed H3Mab‑17 had strongly induced both ADCC and CDC against Caco‑2 cells. In vivo experiments on Caco‑2 xenografts revealed that H3Mab‑17 treatment significantly reduced tumor growth compared with the control mouse IgG. These data indicated that H3Mab‑17 could be a promising treatment option for HER3‑expressing colon cancers.