To investigate the potential applicability of mucosal vaccines against mucosa-unrelated pathogens, a non-parenteral vaccination approach was taken as a prophylactic strategy against mosquito-borne Japanese encephalitis virus (JEV). Intranasal (i.n.) immunization with a mouse brain-derived formalin-inactivated JE vaccine induced a robust virus-neutralizing antibody in mice, and this induction was augmented by co-administration with cholera toxin (CT) and pertussis toxin, but not with killed Bordetella pertussis. The antibody response induced by the i.n. administration of the JE vaccine with bacterial toxins was comparable in intensity to that induced by a parenteral immunization regime, and the former was considerably more effective in terms of delayed-type hypersensitivity and local antibody response. In addition, the adjuvant effects of bacterial toxins were much more prominent for the mucosal than the parenteral route. Two other non-invasive routes, oral and transcutaneous administration, were examined, but the i.n. route was by far the most effective. Finally, the vaccine efficacy of a chimeric fusion protein between the B subunit of CT and the JEV envelope protein showed some promise for the development of non-invasive JE vaccine. Our results suggest that the mucosal vaccination approach is feasible for a non-mucosal pathogen such as JEV, but that the adjuvant, carrier molecule, and administration route must be optimized for construction of an effective vaccine platform.