Abstract: Objective To construct recombinant adenovirus Ad/VP1 and investigate the immune effect against coxsackievirus infection in mice. Methods The recombinant adenovirus Ad/VP1 was constructed and packaged. The target protein was verified by Western blot analysis. BALB/c mice were divided into three groups: Ad/VP1 group, Ad group and PBS group. The mice in each group were immunized by intramuscular injection. The titers of sera IgG and neutralizing antibody were detected by ELISA method and trace neutralization assay, respectively. The specific CTL cytotoxic activity was detected by CCK-8 assay. The mice in each group were challenged with lethal dose of coxsackievirus, the titers of the sera virus were titrated and the protection efficacy against coxsackievirus infection were observed. Results The recombinant adenovirus Ad/VP1 was successfully constructed and target protein was expressed. It’s observed that the titers of CVB3 VP1 specific antibody and neutralizing antibody was much higher than those of the other groups(P<0.01), CTL cytotoxicity activities and protection rate of the Ad/VP1 group were also much higher than the other groups(P<0.05), and the titer of sera virus was lower after CVB3 challenged(P<0.05). Conclusions Both the celluar and humoral immune responses and the protection rate in mice could been significantly enhanced by the Ad/VP1.