A monoclonal antibody having a neutralizing activity on HB-EGF is disclosed. The monoclonal antibody of the present invention is preferably an antibody that does not bind to the HB-EGF protein on the cell surface of HB-EGF-expressing cells. Also provided are an anti-cancer agent and a cell proliferation inhibitor, which comprise the monoclonal antibody of the present invention as an active ingredient, and a method of treating cancer, the method comprising administering the monoclonal antibody of the present invention. Cancers that can be treated by the anti-cancer agent of the present invention include pancreatic cancer, liver cancer, esophageal cancer, melanoma, colorectal cancer, gastric cancer, ovarian cancer, bladder cancer, and brain tumors.