The neddylation pathway belongs post-translational modifications and plays important roles in regulating viral infection and replication. To address the relationship of influenza A virus with the neddylation modification pathway, we demonstrate that IAV infection in A549 cells can activate the neddylation modification pathway to increase virus growth and enhance the expression of pro-inflammatory cytokines to increase pathogenicity. The pre-treatment of Nedd8-activating enzyme subunit 1 (NAE1)-specific inhibitor, MLN4924, interferes with Nedd8 conjugation and NF-κB activity. MLN4924 exhibited pronounced antiviral activity against different subtypes of influenza A virus, including classical H1N1 (PR8), H9N2 subtype, and pandemic H1N1 2009 (pdmH1N1) viruses. Through the inhibition of the CRL/NF-κB pathway, MLN4924 could significantly suppress the expression levels of pro-inflammatory cytokines induced by IAVs. These findings suggest that MLN4924 can be developed as a novel antiviral therapy for influenza infection for anti-viral efficacy and anti-inflammation activity