Background: Non-structural protein 1, a highly conserved influenza virus protein, has been demonstrated previously to be a potential target for antiviral development.Several benzamide derivatives have been identified that inhibit NS1 function specifically and suppress virus replication.But the inhibition mechanism of these inhjbitors in NS 1 is unclear.In this paper, the binding site of these inhibitors to NS1 was explored.Methods: Potential ligand binding sites were predicted by Q-SiteFider and blind docking of JJ3297 (a representative small molecule of benzamide derivatives) to NS 1.In order to find the inhibitor binding site, JJ3297 was submitted to docking simulations, in which JJ3297 were docked into each predicted binding sites of each NS 1 3-D structures that retrieved from PDB.Meanwhile, a series of benzamide derivatives were docked into five predicted binding sites used 2Z0A as NS 1 3-D structure.In order to identify the key features in the interaction between NS1 and ligands, molecular dynamics simulation and MM-GBSA calculation were performed on JJ3297-NS1 complex.Results: Based on results of serveral docking studies on different NS 1 3-D structures and different inhibitors, we found that these inhibitors bound with high affinity at a site located at RNA-binding domain.The predicted binding site is a conserved hydrophobic pocket that allow inhibitors to take part in van der Waals interactions with F14, W16, Q40, L43, I54, T58 and K62 via its hydrophobic group, while the phenol hydroxyl and linking region acylamino group has formed several hydrogen bonding interactions with H 17 and E55.From the MM-GBSA calculations, we find that the van der Waals interaction term provide the main driving force for the binding process, indicating that the hydrophobic groups are crucial for the inhibitory activity.Conclusions: The molecular docking and MM/PBSA method were combinational used to confirm the reasonable binding site of some small molecule inhibitors to NS1.The uncovering of the binding site of these inhibitors to NS 1 will provide a guideline for rational design of anti-influenza virus drug .