Influenza A viruses (IAVs) rely on viral ribonucleoprotein (vRNP) complexes to control transcription and replication. Each vRNP consists of one viral genomic RNA segment associated with multiple nucleoproteins (NP) and a trimeric IAV RNA polymerase complex. Previous studies showed that post‐translational modifications of vRNP components, such as NP, by host factors would in turn affect the IAV life cycle or modulate host anti‐viral response. In this study, we found host E3 ubiquitin ligase Pirh2 interacted with NP and mediated short‐chain ubiquitination of NP at lysine 351, which suppressed NP‐PB2 interaction and vRNP formation. In addition, we showed that knockdown of Pirh2 promoted IAV replication, whereas overexpression of Pirh2 inhibited IAV replication. However, Pirh2‐ΔRING lacking E3 ligase activity failed to inhibit IAV infection. Moreover, we showed that Pirh2 had no effect on the replication of a rescued virus, WSN‐K351R, carrying lysine‐to‐arginine substitution at residue 351. Interestingly, by analyzing human and avian IAVs from 2011 to 2020 in influenza research databases, we found that 99.18% of 26 977 human IAVs encode lysine, but 95.3% of 9956 avian IAVs encode arginine at residue 351 of NP protein. Consistently, knockdown of Pirh2 failed to promote propagation of two avian‐like influenza viruses, H9N2‐W1 and H9N2‐C1, which naturally encode arginine at residue 351 of NP. Taken together, we demonstrated that Pirh2 is a host factor restricting IAV infection by modulating short‐chain ubiquitination of NP. Meanwhile, it is noteworthy that residue 351 of NP targeted by Pirh2 may associate with the evasion of human anti‐viral response against avian‐like influenza viruses. [ABSTRACT FROM AUTHOR]