The H5N6 avian influenza virus, a highly pathogenic strain, poses a significant threat to poultry production and public health. The RNA-dependent RNA polymerase (comprising PB1, PB2, and PA proteins) and nucleoprotein of highly pathogenic avian influenza viruses interact with avian host proteins, influencing the efficiency of viral RNA synthesis and severity of infection. To comprehensively understand how H5N6 avian influenza virus interfaces with host cellular mechanisms during infection and replication, it is essential to identify which host proteins physically associate with viral polymerase proteins. In this study, affinity purification mass spectrometry was used to identify physical interactions between H5N6-JX polymerase proteins (PB1, PB2, PA, and nucleoprotein) and host proteins in chicken DF-1 cells. We identified 455 H5N6-chicken interacting proteins and successfully cloned 231 of these genes. Overexpression experiments revealed several host proteins involved in viral replication in DF-1 cells. Specifically, nine host genes were found to promote avian influenza virus proliferation, whereas 20 inhibited it. Furthermore, we demonstrated that avian NUP93 interacts with the viral PB1 protein, enhancing polymerase transcriptional activity and promoting viral proliferation. These findings provide a more comprehensive understanding of how host mechanisms are manipulated during H5N6 avian influenza viral infection and replication, providing insights into the mechanisms of avian influenza virus cross-species transmission.