To examine the immunogenicity of the plasmid DNA encoding human sperm associated antigen 9 (hSPAG9), the cDNA corresponding to hSPAG9 was cloned in mammalian expression vector pcDNA 3.1 down stream of cytomegalovirus promoter. Immunization of female BALB/cJ mice with pcDNA-hSPAG9 plasmid DNA in saline by intramuscular (i.m.), by adsorbing onto gold microcarriers (delivered by gene gun) and by recombinant hSPAG9 (r-hSPAG9) protein generated antibody response against Escherichia coli expressed r-hSPAG9 protein and native SPAG9 in human sperm. Although mice immunized with r-hSPAG9 protein exhibited highest antibodies titres (P<0.001), the difference in the antibody titres seen by the two modes of plasmid DNA delivery were not significant (P>0.05). A dominant IgG1 isotype response was observed in mice immunized with pcDNA-hSPAG9 plasmid DNA delivered by gene gun as compared to a mixed IgG1-IgG2a isotype response in mice immunized with r-hSPAG9 protein and pcDNA-hSPAG9 plasmid DNA delivered by i.m. Further, antibodies generated by pcDNA-hSPAG9 plasmid DNA localized acrosomal compartment of human sperm and inhibited sperm adherence to or penetration in zona-free hamster egg penetration test. These studies for the first time, demonstrate the feasibility of generating an immune response to sperm specific hSPAG9 protein by DNA vaccine and that antibodies thus generated recognize native SPAG9 in human sperm.