Fusarium graminearum virus-DK21 (FgV-DK21), which infects the plant pathogenic F. graminearum, perturbs host developmental processes such as sporulation, morphology, pigmentation, and attenuates the virulence (hypovirulence) of the host. To identify the differentially expressed F. graminearum proteins by FgV-DK21 infection, we have used two-dimensional electrophoresis with mass spectrometry using proteins extracted from virus-free and FgV-DK21-infected strains. A total of 148 spots showing an altered expression were identified by PDQuest program. Among these spots, 33 spots were exclusively analyzed including 14 spots from FgV-DK21-infected and 19 spots from virus-free strains by ESI-MS/MS analyses and successfully identified 23 proteins. Seven proteins including sporulation-specific gene SPS2, triose phosphate isomerase, nucleoside diphosphate kinase, and woronin body major protein precursor were induced or significantly up-regulated by FgV-DK21 infection. A significant decrease or down regulation of 16 proteins including enolase, saccharopine dehydrogenase, flavohemoglobin, mannitol dehydrogenase and malate dehydrogenase caused by FgV-DK21 infection was also identified. Variations of protein expression were also further investigated at the mRNA level by real-time RT-PCR analysis, which confirmed the proteomic data for 9 out of the representative 11 selected proteins including 5 proteins from up-regulated group and 6 proteins from down-regulated group. Further investigation of these differentially expressed proteins will provide novel insights into the molecular responses of F. graminearum to FgV-DK21 infection.