To identify the neutralizing B-cell epitope of epididymal protease inhibitor (Eppin) and promote its immunogenicity, we designed four candidate B-cell epitopes peptide and employed a recombinant human Eppin (rhEppin) protein prime/peptide boost strategy to compare the immune responses and fertility inhibition effect with that of rhEppin or peptide alone vaccination. Our results indicate that priming with Eppin and boosting with different peptide showed similarly enhanced antibodies titers but different fertility inhibition effect, which coordinated with the motility inhibition of the antisera to human sperm. Thus we explored an alternative approach to induce dominant neutralizing B-cell epitope specific immune response and an ideal protocol for providing effective and long-term fertility inhibition of male mice.