Hyphal fusion (anastomosis) is a common process serving many important functions at various developmental stages in the life cycle of ascomycetous fungi.  However, the biological roles and molecular mechanisms in plant pathogenic fungi were widely unknown.  In this study, a hyphal fusion protein FpHam-2 was screened from a T-DNA insertion mutant library of Fusarium pseudograminearum, and FpHam-2 interacts with another 2 hyphal fusion protein homologues FpHam-3 and FpHam-4.  Each of these 3 genes deletion mutant revealed in similar defective phenotypes compared with the WT and complemented strains, including reduction in growth rate, defects in hyphal fusion and conidiation, more sensitive for cell membrane, cell wall and oxidative stress responses, and decreased in virulence.  The yeast two-hybrid assay was used to identify that FpHam-2 interacts with 3 autophagy-related proteins, including FpAtg3, FpAtg28 and FpAtg33.  Furthermore, FpHam-2-deletion mutant showed decreased accumulation of autophagic bodies in hypha.  In conclusion, FpHam-2, FpHam-3 and FpHam-4 have an essential role for hyphal fusion and regulating the growth, conidiation and virulence in F. pseudograminearum.