In this study, we observed a global increase in intracellular linear ubiquitination upon RNA viral infection, suggesting that linear ubiquitination may be a prerequisite for the initiation of antiviral innate immunity. Furthermore, using a combination of various cell lines and in vivo experiments, we identified OTULIN as a critical negative regulator of RNA virus-induced type I IFN signaling by specifically removing linear ubiquitin chains from TRAF6.