Substances outside the cell are transported into the cell through the deformation movement of the plasma membrane endocytosed by the fungus. Studies on endocytosis-related proteins and their characteristics in plant pathogenic filamentous fungi are still rare. The NPFxD-related proteins that exert endocytosis in plant pathogenic filamentous fungi have not been reported yet. Based on previous studies, and endocytosis keywords were used for searching in the NCBI database. At the same time, based on the sequence of the NPFxD motif protein in the model organism Aspergillus nidulans, online comparison analysis was carried out through Blastp. It is clear that there are 6 proteins with NPFxD motif in Colletotrichum graminicola, which are named CgTPO1, CgMYO2, CgCRN3, CgNPF4, CgGBP5 and CgTRM6 according to the homology relationship. In addition, the NPFxD protein has been analyzed in bioinformatics in terms of transmembrane, conserved domains, subcellular localization, physical and chemical properties, signal peptide, and secondary structure. The results show that the results of different transmembrane prediction software are not the same. There are many types of NPFxD protein conserved domains and lack of the same type of domains. The subcellular location of NPFxD protein is diverse and not the same. Gly and Ala amino acid residues account for the highest proportion and only CgNPF4 has an obvious signal peptide in NPFxD protein. Also, The CgMYO2 protein has a relatively high proportion of alpha helix structure. In addition, the genetic relationship analysis showed that the NPFxD protein of C. graminicola and the bacteria of the genus Colletotrichum had high homology and close genetic relationship. This research lays a solid theoretical foundation for further research on the function of NPFxD motif protein. At the same time, it also provides important theoretical guidance for further research on other Colletotrichum sp..