Lysine acetylation, a form of protein post-translational modifications (PTM), is known to play crucial roles in regulating cellular and physiological processes. In this study, for the first time, lysine acetylation sites were identified on the hemagglutinin (HA) protein of H1N1 subtype influenza virus using mass spectrometry analysis, including K157, K169, K418, and K459. To elucidate the functional significance of these acetylation, eight mutant viruses featuring arginine substitutions (K157R, K169R, K418R, or K459R) to mimic deacetylated states and glutamine substitutions (K157Q, K169Q, K418Q, or K459Q) to mimic constitutively acetylated states were generated by reverse genetics. In mouse model, deacetylation mutants exhibited significantly attenuated virulence compared to wild-type. Interestingly, while the acetylation-mimetic K157Q and K418Q restored viral virulence, the K169Q and K459Q mutants failed to restore the pathogenicity. These findings suggest that acetylation at K157Q and K418Q and dynamical acetylation at residues K169 and K459 are essential for the virulence of influenza virus. Subsequent investigations revealed that acetylation plays a pivotal role in the pH stability of HA protein, mediating viral pathogenicity. Given the critical role of HA acetylation in viral virulence, we investigated the potential of avirulent viruses K157R, K169Q, or K418R as promising live-attenuated vaccine candidates. Notably, the K418R mutant, caused no detectable weight loss, was particularly safe and provided full protection against wild-type influenza virus challenge in mouse model. This study highlights the critical role of HA acetylation modification in influenza virus pathogenicity and offers a new strategy into the development of influenza virus vaccine.