Nutrient and stress factor 1 (Nsf1), a transcription factor containing the classical Cys2-His2 (C2H2) zinc finger motif, is expressed under non-fermentable carbon conditions and in response to salt stress in Saccharomyces cerevisiae. However, the role of Nsf1 in filamentous fungi is not well understood. In this study, the orthologue of Nsf1 was investigated in Fusarium graminearum (named FgNsf1), a causal agent of Fusarium head blight (FHB). The functions of FgNsf1 were evaluated by constructing a FgNSF1 deletion mutant, designated as ΔFgNsf1, and its functional complementation mutant ΔFgNsf1-C. Gene deletion experiments showed that the mycelial growth rate, asexual and sexual reproduction of ΔFgNsf1 were significantly reduced, but the pigment production of ΔFgNsf1 was remarkably increased compared with the PH-1 and ΔFgNsf1-C. In addition, the tolerance of ΔFgNsf1 to osmotic pressures, cell wall-damaging agents and oxidative stress increased significantly. Sensitivity tests to different fungicides revealed that ΔFgNsf1 exhibited increased sensitivity to carbendazim (MBC) and tebuconazole, and enhanced tolerance to fludioxonil and iprodione than PH-1 and ΔFgNsf1-C. The virulence of ΔFgNsf1 to wheat coleoptiles and flowering wheat heads were dramatically decreased, which was consistent with the decrease in the yield of deoxynivalenol (DON). All of these defects were restored by target gene complementation. These results indicated that FgNsf1 plays a crucial role in vegetative growth, asexual and sexual reproduction, stress responses, fungicide sensitivity, and full virulence in F. graminearum.