H5N1 influenza A virus (IAV) causes seasonal epidemics that represent a worldwide threat to public health. IAV relies on host factors for viral replication. PB1 N40 is translated from the fifth starting code (AUG) of PB1 mRNA, which is the product of the ribosomal scan omission. Here, we report the interactome landscape of H5N1 IAV PB1 N40 protein in chicken cells. The interacting complexes were captured by co-immunoprecipitation and analyzed by mass spectrometry. We identified 135 proteins as PB1 N40-interacting proteins. GO and Pathway analysis showed that proteins with biological functions such as protein localization and viral transcription and proteins related to signaling pathways of DNA replication and cell cycle were significantly enriched in virus-host interactions, suggesting the potential roles of them in infection with H5N1 IAV. Comparative analysis among H1N1 and H5N1 revealed conservation of the virus-host protein interaction between different subtypes or strains of influenza virus. ARCN1 was identified as a host interacting factor of H5N1 IAV PB1 N40 protein, which is the component of the coatomer. Knockdown of ARCN1 significantly decreased the titer of H5N1 IAV in chicken cells. BIOLOGICAL SIGNIFICANCE: Influenza A virus (IAV) is a great threat to public health and avian production. However, the manner in which avian IAV recruits the host cellular machinery for replication and how the host antagonizes the IAV infection was previously poorly understood. Here we present the viral-host interactome of the H5N1 IAV PB1 N40 protein and reveal its involvement with dozens of important host genes during the course of IAV infection.