Upon invading the cell, the viral RNA is recognized by the RIG-I receptor located in the cytoplasm, causing the RIG-I receptor to be activated. The activated RIG-I receptor transmits downstream antiviral signals by interacting with the adaptor protein VISA located on the mitochondria, leading to the production of type I interferons and crude inflammatory cytokine genes. Although there have been many studies on antiviral signal transduction of RIG-I receptors in recent years, the mechanism of RIG-I-VISA-mediated antiviral regulation is still not fully understood. In this study, we identified SNX5 as a negative regulator of RLR-mediated antiviral signaling. Our results show that overexpression of SNX5 inhibits viral-induced activation of the IFN-beta promoter, ISRE, NF-kappa B, and IRF3, whereas RNAi knockdown of SNX5 expression shows opposite results. We also found that overexpression of SNX5 enhanced RIG-I's K48 ubiquitination and attenuated its K63 ubiquitination, resulting in inhibition of virus-induced RIG-I expression. Besides, further studies show that SNX5 overexpression weakens the interaction between VISA and TRAF2/5. Our findings suggest that SNX5 negatively regulates RLR-mediated antiviral signaling by targeting the RIG-I-VISA signalosome and provide new evidence for the negative regulation of RIG-I-mediated innate immune response mechanisms. (C) 2019 Published by Elsevier Inc.