To improve the mucosal immune responses and protection against coxsackievirus B3 (CVB3), on the basis of our previously prepared chitosan-pVP1 mucosal vaccine, plasmid encoding lymphotactin (LTN), a C family chemokine, was introduced. chi-(pVP1+pLTN) were prepared by conjugating chitosan with the mixture of 50 μg pVP1 and pLTN. Male BALB/c mice were immunized 4 times biweekly. Two weeks after the final immunization, the level of CVB3-specific serum IgG, fecal IgA and mucosal CTL activity were quantified. Meanwhile, mice were challenged with 3 LD50s, serum creatine kinase (CK) activity and histopathological changes in heart tissue were measured. Compared with control groups, immunization with chi-(pVP1+pLTN) significantly increased the levels of CVB3-specific serum IgG and fecal IgA, and enhanced the mucosal CTL activity. The incidence of myocarditis in mice receiving chi-(pVP1+pLTN) was 16.7%, significantly lower than 33.3% in mice receiving chi-(pVP1+pcDNA3.1). Histopathological analysis showed that fewer inflammatory cells under the epicardium in the hearts of co-immunized mice compared with that of control groups. In conclusion, intranasal co-administration of LTN plasmid via chitosan nanoparticle could enhance CVB3 specific mucosal immune responses and provide more efficient protection against CVB3-induced myocarditis.