The type I IFNs are cytokines that elicit host innate immure responses against tumor and viral infections. Production of type I IFNs is known to be mediated by the activation of pattern recognition receptors (PRRs). During virus infection, virus-derived nucleic acids are mainly sensed by certain PRRs. Particularly, RIG-I is a key PRR that can detect virus-derived RNAs in the cytoplasm during infection, which are closely related to human disease pathogenesis. Recently we have reported that RIG-I has the dual function as an HBV sensor, which recognizes pregenomic RNA and activates innate signaling, and as a direct antiviral effector counteracting viral polymerase during HBV infection. Mechanistically, binding of RIG-I to its ligand RNAs activates the downstream signaling pathways via CARD domain in a manner dependent on the adaptor protein mitochondrial antiviral signaling protein (MAVS; also known as IPS-1), leading to the induction of IRF-3- and NF-kappaB-dependent gene expression of type I IFNs and inflammatory cytokines. Thus, RIG-I sensing of viral RNA is a crucial process to activate the antiviral innate responses to limit viral replication and the subsequent activation of adaptive immunity. In this study, through an affinity isolation-mass spectrometry approach was taken using RIG-I T55I mutant, which cannnot activate the downstream pathway, we identified an E3 ubiquitin-protein ligase RBX1 as an interacting protein. Interestingly RBX1 have been reported to be associated with cancer and allergic rhinitis. Using siRNA knockdown and recombinant proteins, RBX1 interaction with RIG-I resulted in reduced activity of RIG-I helicase, leading to decreased induction of IFN-β and IL-6. Thus, the interplay of RBX1 with RIG-I inhibits the RIG-I signaling, suggesting that RBX1 may function as a negative inhibitor in RIG-I-associated antiviral defense.