Currently, there is increasing spillover of highly pathogenic H5N1 avian influenza virus (AIV) to mammals, raising a concern of pandemic threat about this virus. Although the function of PA protein of the influenza virus is well understood, the understanding of how phosphorylation regulates this protein and influenza viral life cycle is still limited. We previously identified PA S225 as the phosphorylation site in the highly pathogenic H5N1 AIV. In this study, we investigated the role of phosphorylation in regulating PA function and viral fitness through dephosphorylation (PA S225A) or continuous phosphorylation (PA S225E)-mimetic mutation of PA S225. Structure analysis revealed that PA S225A or PA S225E mutation had no obvious effect on the structure of PA protein. Replication assay in vitro showed that PA S225A phosphorylation-ablative mutation significantly inhibited virus replication both in mammalian and avian-derived cells, while PA S225E enhanced viral replication in these cells. Correspondingly, PA S225A dephosphorylation significantly attenuated viral replication and virulence in mice, while PA S225E enhanced these aspects in mice. Mechanistically, PA S225A mutation significantly decreased viral polymerase activity, disabled viral ribonucleoprotein complex (vRNP) assembly and attenuated PA nuclear accumulation. Altogether, our study directly suggested that phosphorylation of PA protein at site S225 enhances viral fitness of the highly pathogenic H5N1 virus in mammals by assuring effective vRNP activity, providing a framework for further study of phosphorylation events in influenza virus life cycle.