Vaccines based on gamete specific proteins have been proposed for fertility inhibition. In the present study, immunogenicity and contraceptive potential of E. coli-expressed recombinant fusion protein TT-KK-ZP3-GGG-Iz, comprising promiscuous T cell epitope of tetanus toxoid (TT) followed by dilysin linker (KK), a fragment of dog zona pellucida glycoprotein-3 (ZP3), triglycine spacer (GGG) and a fragment of dog Izumo (Iz) without any affinity tag has been evaluated in female FvB/J mice. In addition, recombinant TT-KK-ZP3 and Izumo linked to the promiscuous T cell epitope of bovine RNase (bRNase-KK-Iz) and their physical mixture were also used. SDS-PAGE and immunoblot studies revealed ∼32 kDa band corresponding to TT-KK-ZP3-GGG-Iz, ∼22 kDa band of TT-KK-ZP3 and ∼11 kDa band of bRNase-KK-Iz. Groups of mice immunized with the above recombinant proteins led to the generation of high antibody titres against the respective proteins. Immunization with recombinant TT-KK-ZP3-GGG-Iz generated higher antibody titre as compared to mice immunized with physical mixture of TT-KK-ZP3 and bRNase-KK-Iz. Antibodies against TT-KK-ZP3-GGG-Iz and TT-KK-ZP3 recognized mouse and dog ZP and those against TT-KK-ZP3-GGG-Iz and bRNase-KK-Iz recognized mouse and dog acrosome-reacted sperm in an indirect immunofluorescence assay. Immune sera from groups of mice immunized with the above recombinant proteins led to a significant reduction in mouse in vitro fertilization. Mating studies revealed significant reduction in fertility as compared to adjuvant control group. Highest infertility was observed in group of mice immunized with TT-KK-ZP3 followed by TT-KK-ZP3-GGG-Iz. Infertility was associated with the antibody titres against ZP3, whereas no association in the inhibition of fertility and antibody titres against Izumo was observed. In conclusion, these studies revealed the contraceptive potential of ZP3, which could not be further enhanced by the inclusion of Izumo.