[Objective] To study the effect of amino acid mutation(s) in the G-H loop of VP3 of foot-and-mouth disease virus (FMDV) serotype O on its biological characteristics. [Methods] Two site-directed mutants, rHN^V3174Y and rHN^{D3173N+V3174E+N3179C}, were generated by using reverse genetic technique platform of FMDV. The phenotypic properties of the resulting viruses were characterized by plaque forming assays, one-step growth curves, determination of TCID₅₀ and LD₅₀, indirect immunofluorescence assays and laser confocal microscopies. [Results] There were no significant differences in the infectivity, plaque phenotype, and replication kinetics between rHN, rHN^V3174Y and rHN^{D3173N+V3174E+N3179C} for BHK-21 cells. Compared to the backbone virus rHN, however, the pathogenicity of rHN^V3174Y and rHN^{D3173N+V3174E+N3179C} was obvious decreased in suckling mice, and these two rescued viruses gained the ability to infect CHO-K1 cells via caveola-mediated endocytosis. [Conclusion] Individual amino acid mutations at position 3174 in VP3 have an influence on the virulence and endocytic pathway of FMDV serotype O in the infection of host cells, which may help us understand the potentially important functional roles of the G-H loop of VP3 in the 3D spatial conformation of FMDV virion.