[Objective] To successfully establish the C57BL/6 mice experimental infection model for foot-and-mouth disease virus. [Methods] The foot-and-mouth disease virus O/HK/CHA/99 MF4 strain, which is insensitive to C57BL/6 mice, was selected and continuously passaged in C57BL/6 mice (in vivo) and fetal pig kidney cells (in vitro). [Results] We successfully obtained a foot-and-mouth disease virus mutant (O/HK/CHA/99 MF4C5), which was sensitive to C57BL/6 mouse. [Conclusion] This study successfully established C57BL/6 mice model for a foot-and-mouth disease virus mutant strain, which lays a foundation for the evaluation of the efficacy of foot-and-mouth disease vaccine and the study on pathogenicity in the future.