Homologous recombination (HR) and nonhomologous end joining (NHEJ) are considered the two main double-strand break (DSB) repair approaches in eukaryotes. Inhibiting the activities of the key component in NHEJ commonly enhances the efficiency of targeted gene knockouts or affects growth and development in higher eukaryotes. However, little is known about the roles of the NHEJ pathway in foliar pathogens. Here we identified a gene designated StKU80, which encodes a putative DNA end-binding protein homologous to yeast Ku80, in the foliar pathogen Exserohilum turcicum. Conserved domain analysis showed that the typical domains VWA, Ku78 and Ku-PK-bind are usually present in Ku70/80 proteins in eukaryotes and are also present in StKu80. Phylogenetic analysis indicated that StKu80 is most closely related to Ku80 (XP_001802136.1) from Parastagonospora nodorum, followed by Ku80 (AGF90044.1) from Monascus ruber. Furthermore, the gene knockout mutants ΔStKU80-1 and ΔStKU80-2 were obtained. These mutants displayed longer septas, thinner cell walls, smaller amounts of substances on cell wall surfaces, and more mitochondria per cell than the wild-type (WT) strain but similar HT-toxin activity. The mutants did not produce conidia and mature appressoria. On the other hand, the mutants were highly sensitive to H2O2, but not to ultraviolet radiation. In summary, the StKU80 plays devious roles in regulating the development of E. turcicum.