ABSTRACT In this study, we sought to determine whether intratypic and intertypic cross-reactivity protected against enterovirus 71 (EV71) infection in a murine infection model. We demonstrate that active immunization of 1-day-old mice with avirulent EV71 strain or coxsackie A16 virus (CA16) by the oral route developed anti-EV71 antibodies with neutralizing activity (1:16 and 1:2, respectively). Splenocytes from both EV71- and CA16-immunized mice proliferated upon EV71 or CA16, but not coxsackie B3 virus (CB3), antigen stimulation. Immunized mice became more resistant to virulent EV71 strain challenge than nonimmunized mice. There was an increase in the percentage of activated splenic T cells and B cells in the immunized mice 2 days after EV71 challenge. The CA16 immune serum reacted with EV71 antigens in an enzyme-linked immunosorbent assay and neutralized EV71 but not CB3 or poliovirus at a titer of 1:4. Passive immunization with the CA16 immune serum reduced the clinical score, diminished the organ viral load, and increased the survival rate of mice upon EV71 challenge. CB3 neither shared in vitro cross-reactivity with EV71 nor provided in vivo protection after both active and passive immunization. These results illustrated that live vaccine is feasible for EV71 and that intertypic cross-reactivity of enteroviruses may provide a way to determine the prevalence of EV71.