For influenza A viruses (IAVs), non-structural protein 1 (NS1) protein was recognized to be the key factor to enhance virulence by antagonizing host innate anti-viral responses. However, for the pathways allowing NS1 to regulate the type I interferon (IFN) response, the identification of the substrates was still incomplete. Here a recombinant IAV encoding a NS1 containing an affinity tag (NS1-Strep) was generated to capture the NS1-interactome in the lungs of infected mice. Several scaffold proteins of the 14-3-3 family were distinguished as the most potent candidates. Based on the conserved motif RxxTxxT of NS1, the interaction between NS1 and 14-3-3ε was enabled, which competed for the binding of RIG-I to 14-3-3ε and prevented RIG-I translocation to the adaptor MAVS, consequently inhibiting IFN-β expression. A recombinant mutant IAV deficient in 14-3-3ε binding elicited a markable innate immune responses and showed impaired growth kinetics.