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XPG/RAD2 nuclease family plays a crucial role in DNA damage repair to maintain genomic integrity. However, the biological function Mkt1, a member of XPG/RAD2 nuclease family, is still unclear in <i>M. oryzae</i>. Here, we identified and characterized the biological functions of MoMkt1. Our results demonstrated that MoMkt1<i> </i>is involved in hyphal growth, conidiation, normal appressorium development, degradation of glycogen and lipid droplets, responses to cell wall stress and pathogenicity in <i>M. oryzae</i>. Additionally, MoMkt1 is required for scavenging host-derived ROS and response to DNA replicative stress. Further investigation revealed that MoMkt1 interacts with the TFIIH core subunit MoTfb2. Overexpression of <i>MoTFB2</i> slightly affected virulence of <i>M. oryzae</i>. Transcriptome data revealed that <i>MoMKT1</i> regulates metabolic pathways, cell wall, oxidation-reduction process and DNA repair. Our study provides insights into the MoMkt1-mediated development and pathogenicity in <i>M. oryzae</i>.