Retinoic acid-inducible gene I (RIG-I) has recently been identified as one of the key intracellular sensors of virus infection. RIG-I binds to cytosolic double-stranded RNA and initiates a signaling cascade that leads to the activation of transcription factors required for expression of type I interferon (IFN-I). Previous evidence suggests that nonstructural protein 1 (NS1) encoded by influenza A virus (IAV) suppresses IFN-I secretion in virus-infected cells by an unknown mechanism. In the present study, we demonstrate that RIG-I is required for induction of IFN-I in an IAV-infected human lung epithelial cell line. Knockdown of RIG-I expression by RNA interference greatly impairs production of IFN-β in cells infected with different strains of wild-type IAV. Furthermore, co-expression of IAV NS1 down-regulates production of IFN-β induced by RIG-I agonists, and ectopic expression of RIG-I inhibits the replication of IAV. These results provide further information on the mechanism by which IAV NS1 antagonizes the host antiviral response.