Coxsackievirus B3 infections are common causes of acute and chronic myocarditis with no effective prophylactic treatment available. We describe here a prophylactic strategy using chitosan-DNA intranasal immunization to induce CVB3 specific immune responses. Intranasal administration with chitosan-DNA complex prepared by votexing DNA with chitosan, a natural mucus absorption enhancer, resulted in transgenic DNA expression in mouse nasopharynx. Mice immunized with chitosan-DNA (pcDNA3-VP1) encoding VP1, major structural protein of CVB3, produced much higher levels of serum IgG and mucosal secretory IgA compared to mice treated with pcDNA3-VP1 or pcDNA3. Increased virus-specific cytotoxic activity of spleen cells derived from chitosan-DNA vaccinated mice was also determined. Chitosan-pcDNA3-VP1 intranasal immunization resulted in 42.9% protection of mice against lethal CVB3 challenge and a significant reduction of viral load after acute CVB3 infection. Meanwhile no myonecrosis or infiltrating immune cells indicating ongoing myocarditis was detected in hearts of surviving mice treated with chitosan-DNA. Together, Our data show that intranasal delivery of chitosan-DNA vaccine successfully induced mucosal SIgA secretion and might be a promising vaccine candidate to protect against CVB3 infection.