Zona pellucida C (ZPC) is a major glycoprotein of the zona pellucida that possesses the sperm receptor function. ZPC induces autoantibody that can block sperm/oocyte interaction. We selected the partial sequence of rabbit ZPC (amino acid 263–415, rZPC′) as the target and constructed the pCMV4-rZPC′ gene vaccine by using DNA recombinant techniques. The total RNA was extracted from the ovaries of the sexually healthy female rabbit, and the rZPC′ cDNA, which was amplified by reverse transcription-polymerase chain reaction, was directly inserted into the cloning vector PCR2.1 to construct the PCR2.1-rZPC′. This insertion fragment was subcloned into the pCMV4 vector to form the pCMV4-rZPC′ prototype DNA vaccine. All experimental BALB/C mice and New Zealand rabbits received i.m. injection of pCMV4-rZPC′ vaccine three times. The results show that 1) the pCMV4-rZPC′ construct expresses rZPC′ cDNA in mice muscle cells, 2) 60% of the immunized female mice were infertile at 6 wk after the immunization, 3) the mice immunized with pCMV4-rZPC′ DNA vaccine developed anti-rZPC antibodies that bound to the ovarian ZP in situ, and 4) antibodies against rZPC′ were also bound to normal animal ovarian ZP in vitro. The results indicate that anti-rZPC antibodies developed from pCMV4-rZPC′ DNA vaccine can prevent the fertility course and do not interfere with normal follicular development. The pCMV4-rZPC′ DNA vaccine may be possible to develop as a contraceptive vaccine.