Under RNA virus infection, retinoic acid-inducible gene I (RIG-I) in host cells recognizes viral RNA and activates the expression of type I IFN. To investigate the roles of protein methyltransferases and demethylases in RIG-I antiviral signaling pathway, we screened all the known related enzymes with a siRNA library and identified LSD1 as a positive regulator for RIG-I signaling. Exogenous expression of LSD1 enhances RIG-I signaling activated by virus stimulation, whereas its deficiency restricts it. LSD1 interacts with RIG-I, promotes its K63-linked polyubiquitination and interaction with VISA/MAVS. Interestingly, LSD1 exerts its function in antiviral response not dependent on its demethylase activity but through enhancing the interaction between RIG-I with E3 ligases, especially TRIM25. Furthermore, we provide in vivo evidence that LSD1 increases antiviral gene expression and inhibits viral replication. Taken together, our findings demonstrate that LSD1 is a positive regulator of signaling pathway triggered by RNA-virus through mediating RIG-I polyubiquitination.