Nucleoside diphosphate kinase (NDK) plays an important role in many cellular processes in all organisms. In this study, we functionally characterized a nucleoside diphosphate kinase (FgNdk1) in Fusarium graminearum, a causal agent of Fusarium head blight (FHB). FgNdk1 was involved in the generation of energy in the electron-transfer chain by interacting with succinate dehydrogenase (FgSdhA, FgSdhC 1 , and FgSdhC 2 ). Deletion of FgNdk1 not only resulted in abnormal mitochondrial morphology, decreased ATP content, defective fungal development, and impairment in the formation of the toxisome but also led to the suppressed expression level of DON biosynthesis enzymes, decreased DON biosynthesis, and declined pathogenicity as well. Furthermore, deletion of FgNdk1 caused increasing transcriptional levels of FgSdhC 1 and FgdhC 2 , in the presence of pydiflumetofen, related to the decreased sensitivity to SDHI fungicides. Overall, this study identified a new regulatory mechanism of FgNdk1 in the pathogenicity and SDHI fungicide sensitivity of Fusarium graminearum.