The PA gene of the 2009 pandemic H1N1 (pdm09/H1N1) lineage is derived from avian influenza virus (AIV). Together with other polymerase subunits, it enhances the adaptation of avian-origin influenza virus to mammals. However, the functional region of the PA protein remains unclear. Using reverse genetics, we mapped functional domains of pdm09/H1N1 PA in an avian-origin H9N2 canine influenza virus (CIV) background. The N-terminal 169-252 region conferred high intrinsic polymerase activity yet failed to support efficient viral replication alone, revealing a dissociation between catalytic potential and replicative fitness. In contrast, the 85-168 region enabled robust replication despite lower activity. Importantly, synergy between the 169-252 region and its cognate pdm09/H1N1 NP drove high polymerase activity and replication. This pairing also enhanced viral growth in vitro, increased viral titers in mouse lungs and nasal turbinate, and induced pulmonary damage. Mechanistically, the 169-252 region interacts with NP, accelerating nuclear import and cRNA synthesis, thereby optimizing viral RNA replication timing. Thus, PA-NP co-evolution is a key driver of mammalian adaptation, moving beyond single mutations and highlighting internal gene compatibility as a determinant of viral fitness and pandemic potential.