NQO1 (NAD(P)H:quinone oxidoreductase 1) is a well-known antioxidant protein which has enzymatic activity related to chemoprotection of ROS induced damages. Although NQO1 is involved in several part of immune response or cancer prevention, its role in viral infection has never been investigated. In this study, we showed the role of NQOI as a negative regulator of antiviral immune response. Knockdown of FAF1 in several cell lines or knockout in BMDM cells derived from NQOI-KO mice markedly enhances resistance to RNA virus infection and shows higher level of inflammatory cytokines and type I interferon production. Controversially, Overexpression of NQOI in various cell types reduced the production of inflammatory cytokines and Type I interferons upon virus infection. Consistent with in vitro results, upon infection of Vesicular Stomatitis virus (Indiana strain), NQOI-KO mice exhibited marked resistance to virus infection and increased level of IFN-β and IL-6 from serum. These findings suggest that NQOI is a negative regulator that modulates the NF-κB and Type I interferon signaling pathway against virus infection. [The National Research Foundation of Korea (Grant no. 2015020957)]