Protein glycosylation, a co- and post-translational modification that enhances the functional diversity of the proteome, contributes to various molecular and cellular functions by transferring different polysaccharides onto proteins. During the last decade, the role of glycosylation in plant pathogenic fungi has received significant attention, and glycoproteins are expected to play essential roles in various biological processes including pathogenicity. However, the comprehensive functional genetic analyses for protein glycosylation pathways and glycan structures of phytopathogenic fungi are still largely unknown. Here, we investigated the role of protein glycosylation in F. graminearum by identifying 74 genes putative genes involved in the protein glycosylation pathway and characterizing their functions, with a focus on glycan structures. Quantitative proteomics analysis revealed that two key players in the initial core N-glycosylation pathway, Alg3 and Alg12, regulate a wide range of glycoproteins, influencing protein functions and ultimately impacting the virulence of F. graminearum. This study elucidates the complex roles of glycosylation, highlighting the connections among genes involved in the protein glycosylation pathway, glycans, and glycoproteins in regulating the general biology and pathogenicity of Fusarium graminearum. It also would be the fungal glycoproteome study initiative.