Background: Fusarium crown rot a major disease in wheat. However, wheat defense mechanisms remain poorly understood.Results: In this study, we employed tandem mass tag (TMT) quantitative proteomics technology for one disease-susceptible (UC1110 (S1)) and one disease-tolerant wheat cultivar (PI610750 (S2)) inoculated with Fusarium pseudograminearum WZ-8A. Analysis of morphology and physiology showed that average seedling root diameter was significantly decreased 3 days post-inoculation (dpi) in both cultivars. Malondialdehyde content decreased in PI610750 and catalase activity increased in UC1110, indicating that morphology, physiology, and biochemistry differed in both cultivars in response to disease. TMT analysis identified 366 differentially expressed proteins (DEPs) by Gene Ontology enrichment and the Kyoto Encyclopedia of Genes and Genomes in the two comparison groups, S1-3dpi/S1-0dpi (163) and S2-3dpi/S2-0dpi (203). We concluded that phenylpropanoid biosynthesis (8), secondary metabolite biosynthesis (12), linolenic acid metabolites (5), glutathione metabolism (8), plant hormone signal transduction (3), MAPK signaling pathway-plant (4), and photosynthesis (12) contributed to wheat defense mechanisms. Protein-protein interaction network analysis showed that DEPs interacted in sugar metabolism and photosynthesis pathways. We validated 16 genes by real-time quantitative polymerase chain reaction.Conclusion: The results were consistent with proteomics data. Our results provided insight into molecular mechanisms of interaction between wheat and F. pseudograminearum.