Two types of antibodies (35-1 antibody and 292 antibody) capable of binding to phenylalanine at position 115, isoleucine at position 117, glycine at position 140, glutamic acid at position 141, and arginine at position 142 of a human HB-EGF protein were successfully obtained. Then, it was also found that these antibodies had an activity of suppressing cleavage of the human HB-EGF protein, and an activity of suppressing EGFR phosphorylation that would occur when the human HB-EGF bound to the EGFR. Further, determined were amino acid sequences of light chain and heavy chain variable regions of these antibodies and sequences of CDRs of each of the variable regions.