Ubiquitination plays a crucial role in retinoic acid-inducible gene Ⅰ (RIG-Ⅰ)-induced antiviral responses.However,the precise regulatory mechanisms of RIG-Ⅰ activity mediated by conjugated and unanchored ubiquitin chains remain to be determined.In this study,we discovered that T55 of RIG-Ⅰ was required for its binding ability for the unanchored ubiquitin chains.Experimental and mathematical analysis showed that unanchored ubiquitin chains associated with RIG-Ⅰ were essential for sustained activation of type Ⅰ interferon (IFN) signaling.Transcriptomics study revealed that the binding of RIG-I with unanchored ubiquitin chains additionally regulated the expression of a subset of metabolic and cell fate decision genes.Moreover,we found that ubiquitin-specific peptidase 21 (USP21) and USP3 deubiquitinate conjugated and unanchored ubiquitin chains on RIG-I respectively.Taken together,characterization of the regulation mode and functions of conjugated ubiquitination and the unconjugated ubiquitin chainbinding of RIG-I may provide means to fine-tune RIG-Ⅰ-mediated type Ⅰ IFN signaling.