DNA vaccination using a plasmid encoding the rotavirus inner capsid VP6 has been explored in the mouse model of rotavirus infection. BALB/c mice were immunized with a VP6 DNA vaccine by the intramuscular, nasal and oral routes. VP6 DNA vaccination by the nasal and oral routes induced the production of anti-VP6 IgA antibodies by intestinal lymphoid cells. Intramuscular DNA injection stimulated the production of serum anti-VP6 IgG but not serum anti-VP6 IgA antibodies. Protection against shedding of rotaviruses in stools after oral challenge with the murine EDIM rotavirus strain was investigated in the immunized mice. A significant reduction in the level of rotavirus antigen shedding was demonstrated in those mice immunized at mucosal surfaces, both orally and nasally, with the VP6 DNA vaccine. Intramuscular DNA immunization, which elicited serum anti-VP6 IgG responses but not virus-specific intestinal IgA antibodies, did not provide significant protection against rotavirus challenge.