Transmission-blocking vaccines target the sexual stages of the malaria parasite and prevent further development within the mosquito vector halting the transmission of the parasite. Zygote/ookinetes are potential targets of antibodies inhibiting oocyst development in the mosquito midgut and rendering mosquitoes non-infectious. DNA vaccine constructs were developed expressing Pvs25 and Pvs28 (Plasmodium vivax zygote/ookinete surface proteins) fused at the amino terminus with tissue plasminogen activator signal peptide. Antibodies produced in mice after immunization with three doses recognized respective antigens in the parasites and in an ELISA, and these antibodies when tested in membrane feeding assay were potent blockers of P. vivax transmission. Co-immunization with Pvs25 and Pvs28 DNA vaccine constructs did not affect the antigen specific antibody responses against individual antigens, and the antibodies remained effective in blocking parasite transmission demonstrating 91-99% reduction in oocyst number in the mosquito midgut. Several combinations of homologous and heterologous antigen-delivery prime boost strategy were also evaluated and the results suggested that antibody titers and transmission-blocking activities by the three prime-boost strategies (DNA prime/DNA boost, DNA prime/protein boost, and protein prime/protein boost) were comparable with slightly better immunogenicity of heterologous antigen-delivery prime/boost as compared to DNA/DNA alone. These results demonstrate potent immunogenicity of DNA vaccines encoding Pvs25 and Pvs28 and warrant further evaluation in non-human primates.