We previously demonstrated that despite no airborne transmissibility increase compared to low pathogenic avian influenza viruses， select human isolates of highly pathogenic avian influenza A(H7N9) virus exhibit greater virulence in animal models and a lower threshold pH for fusion. In the current study， we utilized both in vitro and in vivo approaches to identify key residues responsible for hemagglutinin (HA) intracellular cleavage， acid stability， and virulence in mice. We found that the four amino acid insertion (-KRTA-) at the HA cleavage site of A/Taiwan/1/2017 virus is essential for HA intracellular cleavage and contributes to disease in mice. Furthermore， a lysine to glutamic acid mutation at position HA2-64 increased the threshold pH for HA activation， reduced virus stability， and replication in mice. Identification of a key residue responsible for enhanced acid stability of A(H7N9) viruses is of great significance for future surveillance activities and improvements in vaccine stability.