Influenza A virus causes epidemics of respiratory diseases in humans leading to thousands of death annually. One of its major virulence factors, the non-structural protein 1 (NS1), exhibits interferon-antagonistic properties. While epithelial cells of the respiratory tract are the primary targets of influenza virus, the virus-sensing mechanisms in these cells eventually leading to IFNbeta production are incompletely understood. Here we show that infection of epithelial cells with NS1-deficient influenza A virus upregulated expression of two molecules that have been previously implicated in sensing of RNA viruses, the retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA5). Gene silencing and overexpression experiments demonstrated that RIG-I, its adapter interferon-beta promoter stimulator 1 (IPS-1) and interferon-regulated factor 3 (IRF3) were involved in influenza A virus-mediated production of the antiviral IFNbeta. In addition, we showed that the NS1 protein is capable to inhibit the RIG-I-induced signalling, a mechanism which corresponded to the observation that only NS1-deficient but not the wild-type virus induced high-level production of IFNbeta. In conclusion, we demonstrated a critical involvement of RIG-I, IPS-1 and IRF3 in influenza A virus infection of epithelial cells.