Polymerase acidic (PA) protein is a multifunctional regulator of influenza A virus (IAV) replication and pathogenesis. In a previous study, we reported that nucleolin (NCL) is a novel PA-interacting host protein. In this study, we further explored the role of NCL during highly pathogenic H5N1 avian influenza virus infection. We found that depletion of endogenous NCL in mammalian cells by siRNA targeting during H5N1 infection resulted in significantly increased viral polymerase activity, elevated viral mRNA, cRNA and vRNA synthesis, accelerated viral replication, and enhanced apoptosis and necrosis. Moreover, siRNA silencing of NCL significantly exacerbated the inflammatory response, resulting in increased secretion of IL-6, TNF-α, TNF-β, CCL-4, CCL-8, IFN-α, IFN-β and IFN-γ. Conversely, overexpression of NCL significantly decreased IAV replication. Collectively, these data show that NCL acts as a novel potential antiviral factor during H5N1 infection. Further studies exploring the antiviral mechanisms of NCL may accelerate the development of new anti-influenza drugs.