NS1 (Non-structural protein 1) is a non-structural protein that can highly express when the avian influenza virus infects the host cells. NS1 can interact with various proteins to alter the intracellular distribution of host proteins and regulate the virulence and pathogenicity of the avian influenza virus. To further study the role of NS1 protein in replication and pathogenesis of avian influenza virus, Glutathione S-transferase (GST) Pull-down was used for screening more proteins interacting with NS1 in human lung adenocarcinoma cell line A549. By mass spectrometry, a potential interacted protein is identified as α-actinin 4 and its interaction with NS1 has not been reported yet. The interaction between NS1 and α-actinin 4 in vitro was confirmed by enzyme-linked immunosorbent assay experiments, and the results showed that the absorbance value of OD450nm in the experimental group was positively correlated with the concentration of NS1-GST protein compared to the negative control group. The co-immunoprecipitation and immunofluorescence results further confirmed the interaction between NS1 and α-actinin 4 at the cellular level. The interaction between NS1 and α-actinin 4 provided a new target for pathogenic mechanism studying and drug screening.