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Grass carp reovirus (GCRV), causative agent of grass carp hemorrhagic disease, causes high mortality of grass carp and results in huge economic losses in grass carp farming. Vaccination is considered the most effective way of preventing GCRV infection and losses of grass carp. In the study, recombination pcDNA3.1-vp4 and pcDNA3.1-vp4-3 plasmids were constructed and respectively transformed into E. coli DH5α. The pcDNA3.1-vp4 and pcDNA3.1-vp4-3 plasmids were used to vaccinate grass carp via intramuscular injection and we evaluated the immune responses induced by different forms of the DNA vaccine and investigated the efficacy in vaccinated fish. The results indicate that better immune responses of grass carp immunized with the pcDNA3.1-vp4 (10 μg/fish) compared with the pcDNA3.1-vp4-3 (10 μg/fish) DNA vaccine. Immune genes were expressed at higher levels in pcDNA3.1-vp4-3 treatment group compared to pcDNA3.1-vp4. The survival post vaccination and challenge of pcDNA3.1-vp4-3 vaccinated fish was 57.89% compared to 36.84% for pcDNA3.1-vp4 group and at 100% mortality in control groups. These results indicated that the segment of vp4-3 could be a plausible candidate for preventing GCRV-JX02 infection among grass carp. • Grass carp injected with pcDNA3.1-vp4 and pcDNA3.1-vp4-3 confers strong immunity against GCRV-JX02. • The DNA vaccine was manufactured by the core of dominant antigen epitope of GCRV-JX02, and its protective effects on grass carp against GCRV-JX02 infection by injection were assessment. • pcDNA3.1-vp4-3 could be a potential therapeutic vaccine against GCRV-JX02. [ABSTRACT FROM AUTHOR]