Peptide mimics of a conformational epitope that is recognized by a mAb with antitumor activity are promising candidates for formulations of anticancer vaccines. These mimotope vaccines are able to induce a polyclonal Ab response focused to the determinant of the mAb. Such attempts at cancer immunotherapy are of special interest for malignant melanoma that is highly resistant to chemotherapy and radiotherapy. In this study, we describe for the first time the design and immunogenicity of a vaccine containing a mimotope of the human high m.w. melanoma-associated Ag (HMW-MAA) and the biological potential of the induced Abs. Mimotopes were selected from a pVIII-9mer phage display peptide library with the anti-HMW-MAA mAb 225.28S. The mimotope vaccine was then generated by coupling the most suitable candidate mimotope to tetanus toxoid as an immunogenic carrier. Immunization of rabbits with this vaccine induced a specific humoral immune response directed toward the epitope recognized by the mAb 225.28S on the native HMW-MAA. The induced Abs inhibited the in vitro growth of the melanoma cell line 518A2 up to 62%. In addition, the Abs mediated 26% lysis of 518A2 cells in Ab-dependent cellular cytotoxicity. Our results indicate a possible application of this mimotope vaccine as a novel immunotherapeutic agent for the treatment of malignant melanoma.