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The human zona pellucida glycoprotein-3 (hZP3) by virtue of its critical role during fertilization has been proposed as a promising candidate antigen to develop a contraceptive vaccine. In this direction, it is imperative to map minimal motifs of the B cell epitopes (BCEs) so as to avoid ZP-specific oophoritogenic T cell epitopes (TCEs) in the ZP3-based immunogens. In this study, based on known results of mapping marmoset and bonnet monkey ZP3 (mstZP3 and bmZP3), two predictableepitopes23–30 and 301–320on hZP3 were first confirmed and five minimal motifs within four epitopes on hZP3 were defined using serum to recombinanthZP3a22–176orhZP3b177–348as well as a biosynthetic peptide strategy. These defined minimal motifs wereQPLWLL23–28forhZP323–30,MQVTDD103–108forhZP393–110,EENW178–181forhZP3172–190, as well asSNSWF306–310andEGP313–315forhZP3301–320, respectively. Furthermore, the antigenicity of two peptides forhZP3172–187andhZP3301–315and specificity of the antibody response to these peptides were also evaluated, which produced high-titer antibodies in immunized animals that were capable of reacting to ZP on human oocytes,r-hZP3b177–348protein, as well asr-hZP3172–190,r-hZP3303–310, andr-hZP3313–320epitope peptides fused with truncated GST188 protein.