To overcome the inability of a conventional vaccine against a virus to induce rapid protection against viral challenge, a novel strategy was performed to generate a novel bi-functional vector expressing antisense RNA targeted to 5' untranslated regions (UTR) and VP1 protein of foot-and-mouth disease virus (FMDV). FMDV 5'UTR containing the viral RNA replication start elements was inserted inversely into the pIRES vector to produce antisense RNA, followed by insertion of FMDV VP1 gene to generate a recombinant plasmid pAS-IR-VP1. BHK-21 cells transfected with pAS-IR-VP1 plasmid showed a specific resistance against FMDV infection. In mice vaccinated with this plasmid, T cell proliferation was significantly higher than that in an unvaccinated control group. Anti-FMDV antibodies were detected up to 1:64 in the serum collected from mice boosted with pAS-IR-VP1 at 21 days after the first immunization. At 6h post-vaccination 50-83% of the suckling mice survived a challenge with FMDV. The results demonstrated that a novel bi-functional DNA vaccine, producing antisense RNA targeted to FMDV 5'UTR and expressing VP1 protein, has been successfully constructed and was able to induce a rapid inhibitory effect and immune response against FMDV infection in mice.