Newcastle disease virus (NDV) is an avian paramyxovirus with oncolytic properties which shows promising effects in the treatment of cancer. Anti-cancer effects are due to the virus ability: i) to replicate in and kill tumor cells, leading finally to their selective elimination; and ii) to induce the stimulation of antitumor activities in immune cells. NDV does not harm normal cells and has a high safety profile. In this study, we first report a direct correlation between the degree of cell resistance to NDV infection and the cellular expression of the retinoic acid-inducible gene I (RIG-I) which is a cytosolic viral RNA receptor. RIG-I plays an important role in the recognition of and response to infection by RNA viruses. We also demonstrate that impairment of the interferon (IFN) pathway through deletion of the receptor for type I IFN (IFNR1) in primary macrophages leads to NDV replication. In tumor cells, addition of exogenous IFN-α4 is shown to lead to tumor growth reduction and inhibition of viral replication. Finally, increase of the RIG-I concentration of tumor cells via plasmid transfection is shown to be associated with a stronger resistance to NDV infection. These findings shed new light on the crucial role played by the cytosolic receptor RIG-I and the plasma membrane receptor IFNR1 as key molecules to protect cells against infection by NDV.