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Monilinia fructicola is the most widely distributed species among the Monilinia genus in the world, and causes blossom blight, twig canker, and fruit rot on Rosaceae fruits. To date, studies on genomics and pathogenicity are limited in M. fructicola. In this study, we identified a redox‐related gene, MfOfd1, which was significantly up‐regulated at 1 hr after inoculation of M. fructicola on peach fruits. We used the clustered regulatory inter‐spaced short palindromic repeats (CRISPR)/Cas9 system combined with homologous recombination to determine the function of the MfOfd1 gene. The results showed that the sporulation of knockdown transformants was reduced by 53% to 83%. The knockdown transformants showed increased sensitivity to H2O2 and decreased virulence on peach fruits compared to the wild‐type isolate Bmpc7. It was found that H2O2 could stimulate the expression of MfOfd1 in the wild‐type isolate. The transformants were also more sensitive to exogenous osmotic stress, such as glycerol, d‐sorbitol, and NaCl, and to dicarboximide fungicides (iprodione and dimethachlon). These results indicate that the MfOfd1 gene plays an important role in M. fructicola in sporulation, oxidative response, osmotic stress tolerance, and virulence. [ABSTRACT FROM AUTHOR]