Objective To determine the feasibility of pcDNA3-1/mESP as a potential immunocontraceptive antigen. Methods Eukaryotic expression plasmid pcDNA3-1/mESP, encoding N-terminal of mouse sperm equatorial segment protein (mESP), was constructed. Two groups of mice（EM>n/EM>=8/group） were inoculated with plasmids of pcDNA3-1/mESP and pcDNA3-1 respectively. RT-PCR， ELISA and double immunofluorescence assay were performed to observe pcDNA3-1/mESP expression and immune response in the inoculated mice. Mouse mating test was employed to study fertility of the experimental and control group. Results mESP cDNA positive band was detected in muscle from mice immunized with pcDNA3-1/mESP，which identified the expression of the recombinant plasmid. The pcDNA3-1/mESP induced specific antibody against mESP in the experimental mice. Significant difference of mean litter size between experimental and control groups was observed, while the rate of fertility did not decrease. Conclusion This study indicates the need to strengthen DNA vaccines in order to obtain