Encephalomyocarditis virus (EMCV) infection can cause acute myocarditis and sudden death in pre-weaned piglets as well as severe reproductive failure in sows. In this study, two recombinant adenoviruses containing capsid precursor polypeptide P1 alone (Ad-P1) and P1 plus nonstructural protein 2A and 3C protease coding regions (Ad-P12A3C) of EMCV were respectively constructed using replication-defective human adenovirus serotype 5 as vector, and their antibody responses and protective efficacies against a lethal EMCV challenge were evaluated in mice. Both Ad-P1 and Ad-P12A3C were confirmed to be capable of expressing VP1 protein in BHK21 cells by immunoperoxidase monolayer assay (IPMA). The results showed that mice vaccinated once or twice with Ad-P1 and Ad-P12A3C generated specific antibody response against VP1 protein of EMCV. Although Ad-P1 induced higher antibody titers, virus-neutralizing antibody response was considerably less (p<0.05), compared to that of Ad-P12A3C. Upon challenging with a virulent EMCV strain, Ad-P12A3C elicited efficacious protection (100% for both vaccination once and twice) in the vaccinated mice; whereas the mice immunized with Ad-P1 showed a lower protection (12.5% for vaccination once and 75% for twice). Our work suggests that the recombinant adenovirus (Ad-P12A3C) containing the capsid precursor polypeptide coding region (P1) plus nonstructural protein 2A and 3C protease genes have an excellent potential to be used as a vaccine that can provide sufficient protective efficacy against EMCV infection in animals.