Avian influenza (AI) significantly threatens poultry health and causes major economic losses in the poultry industry. Vaccination remains crucial for AI prevention and control. The major protective epitopes of influenza viruses are located on hemagglutinin (HA), a surface glycoprotein essential for viral infection. Most influenza vaccines induce neutralizing antibodies against HA to block viral entry. HA maturation requires the HA0 precursor to be proteolytically cleaved at a conserved site by host proteases to yield HA1 and HA2 subunits. A subsequent acidic condition triggers HA conformational changes, enabling viral-host membrane fusion. However, whether HA conformational variations affect immunogenicity remains unclear. In this study, the cleavage site of the HA gene from an H9N2 avian influenza virus was modified to block the proteolytic cleavage of the HA protein. Our results revealed distinct proteolytic patterns of certain mutants, which exhibited either increased or decreased cleavage efficiencies compared to the wild-type (WT) HA. However, none of the mutants exhibited completely abolished HA0 cleavage. To assess the immunogenicity of these variants, BALB/c mice were immunized with DNA vaccines expressing either WT or mutant HA proteins. Strikingly, the mutant HA protein with a 19-amino-acid deletion Dlt5 (P6~P1, P1´~P´13) at the cleavage site exhibited reduced cleavage efficiency and induced significantly higher HI antibody titers compared to the WT. These results offer valuable perspectives for enhancing avian influenza vaccine efficacy through strategic modification of HA cleavage properties.