Magnaporthe oryzae is an important plant-pathogenic fungus of rice that severely endanger the yield and quality of rice. We characterized a F-box protein Mofbox in M. oryzae, a homolog of F-box in Saccharomyces cerevisiae. The F-box possessed typical domains of F-box. The gene knockout vector was transformed into Magnaporthe oryzae to get two gene deletion mutants by principle of homologous recombination. The mutant caused retarded vegetative growth of mycelia, and significantly reduced the conidium production and appressorium formation rate, and the mutant exhibited higher sensitivity to hydrogen peroxide (H₂O₂), and changed cell wall integrity, and could significantly reduce virulence on barley and rice cultivar CO-39.