Newcastle disease, designated a class A disease of poultry by the Office international des epizooties (OIE), is an acute infection caused by Newcastle disease virus (NDV). The merging of the envelope of NDV with the membrane of a target host cell is the key step in the infection pathway, which is driven by the concerted action of two glycoproteins: haemagglutinin- neuraminidase (HN) and fusion (F) protein. When the HN protein binds to the host cell surface receptor, the F protein is activated to mediate fusion. The three-dimensional structure of the F protein has been reported to have low electron density between the DIII domain and the HRB domain, and this electron-poor region is defined as the HRB linker. To clarify the contributing role of the HRB linker in the NDV F protein-mediated fusion process, 6 single amino acid mutants were obtained by site-directed mutagenesis of the HRB linker. The expression of the mutants and their abilities to mediate fusion were analysed, and the key amino acids in the HRB linker were identified as L436, E439, I450, and S453, as they can modulate the fusion ability or expression of the active form to a certain extent. The data shed light on the crucial role of the F protein HRB linker in the acquisition of a normal fusogenic phenotype.