Retinoic acid–inducible gene-I (RIG-I)–like receptors (RLRs) are major cytosolic RNA sensors and play crucial roles in initiating antiviral innate immunity. Furthermore, RLRs have been implicated in multiple autoimmune disorders. Thus, RLR activation should be tightly controlled to avoid detrimental effects. “DEAD-box RNA helicase 3, X-linked” (DDX3X) is a key adaptor in RLR signaling, but its regulatory mechanisms remain unknown. Here, we show that the E3 ubiquitin ligase RNF39 inhibits RLR pathways through mediating K48-linked ubiquitination and proteasomal degradation of DDX3X. Concordantly, Rnf39 deficiency enhances RNA virus–triggered innate immune responses and attenuates viral replication. Thus, our results uncover a previously unknown mechanism for the control of DDX3X activity and suggest RNF39 as a priming intervention target for diseases caused by aberrant RLR activation.