Changes in the NP gene of H5N1 highly pathogenic avian influenza (HPAI) viruses have previously been shown to affect viral replication, alter host gene expression levels and affect mean death times in infected chickens. Five amino acids at positions 22, 184, 400, 406, and 423 were different between the two recombinant viruses studied. In this study, we individually mutated the five amino acids that differed and determined that the difference in virus pathogenicity after NP gene exchange was a result of an alanine to lysine change at position 184 of the NP protein. Infection with viruses containing a lysine at NP 184 induced earlier mortality in chickens, increased virus titers and nitric oxide levels in tissues, and resulted in up-regulated host immune genes, such as alpha-interferon (IFN-alpha), gamma-interferon (IFN-gamma), orthomyxovirus resistance gene 1 (Mx1), and inducible nitric oxide synthase (iNOS). This study underlines the importance of the NP in avian influenza virus replication and pathogenicity.