Objective To construct a DNA contraception vaccine targeting canine zona pellucida 3 (CZP3) antigen and assess the immunological efficacy and contraceptive effect of the vaccine. Methods The CZP3 gene was amplified from total RNA of canine ovary by reverse transcription PCR and analyzed by bioinformatics, such as ProtScale, TMHMM, Signal P, InterProScan, PREDICT PROTEIN, homology modeling, etc. The constructed DNA vaccine pcDNA-CZP3 was used to vaccinate mice, and then its immune effect and contraceptive effect were evaluated in the mice. Results The CZP3 gene had 426 amino acids with two hydrophobic regions at its N-terminal and C-terminal, respectively. The top 22 amino acids at the N-terminal of the CZP3 was the signal peptide and there was a transmembranous helix from extracellular to intracellular at the C-terminal. CZP3 also had 8 B cell epitopes. The DNA contraception vaccine pcDNA3-CZP3 induced high levels of antibody and lower average litter size of mouse compared with the blank and negative control groups significantly. Conclusion The canine contraception DNA vaccine pcDNA-CZP3 has been successfully constructed and it can reduce the mouse fertility remarkably.