The continuous antigenic shift and drift of influenza A virus (IAV) result in the emergence of novel strains and drug resistance. Accordingly, it is urgent to develop novel antiviral drugs that target host factors. Here, we revealed that tyrosine kinase LYN (Lck/Yes-related novel protein tyrosine kinase) interacts directly with nucleoprotein (NP) and reduces the replication and virulence of IAV in vitro and in vivo, in a kinase-dependent manner. By directly catalyzing the tyrosine phosphorylation of NP at Y10/40/97 sites, LYN impairs the interaction of NP with viral RNA and polymerase proteins, as well as the oligomerization of NP, and thus negatively modulates the assembly of the viral ribonucleoprotein complex. The NPY10/40/97F mutation significantly increases the pathogenicity of IAV in mice. Furthermore, the LYN-specific agonist MLR-1023 showed promising therapeutic effects against IAV. Collectively, our findings suggest that LYN is a novel host kinase restricting IAV replication, and a promising target for anti-influenza drug development.