The negative regulation of antiviral immune responses is essential for the host to maintain homeostasis. Jumonji domain-containing protein 6 (JMJD6) was previously identified with a number of functions during RNA virus infection. Upon viral RNA recognition, retinoic acid-inducible gene-I-like receptors (RLRs) physically interact with the mitochondrial antiviral signaling protein (MAVS) and activate TANK-binding kinase 1 (TBK1) to induce type-I interferon (IFN-I) production. Here, JMJD6 was demonstrated to reduce type-I interferon (IFN-I) production in response to cytosolic poly (I:C) and RNA virus infections, including Sendai virus (SeV) and Vesicular stomatitis virus (VSV). Genetic inactivation of JMJD6 enhanced IFN-I production and impaired viral replication. Our unbiased proteomic screen demonstrated JMJD6 contributes to IRF3 K48 ubiquitination degradation in an RNF5-dependent manner. Mice with gene deletion of JMJD6 through piggyBac transposon-mediated gene transfer showed increased VSV-triggered IFN-I production and reduced susceptibility to the virus. These findings classify JMJD6 as a negative regulator of the host's innate immune responses to cytosolic viral RNA. Author summary: RLRs-mediated signaling needs to be terminated in order to prevent persistent immune responses and adverse effects to the host once the virus has been cleared. In this study, we provide rigorous evidence that JMJD6 negatively regulates RLRs-mediated innate immune responses. We found that JMJD6 recruits RNF5 to induce the K48-linked polyubiquitination and proteasomal degradation of activated IRF3. Genetic inactivation of JMJD6 in cells increases IFN-I production to suppress viral infection. Consistently, in vivo studies show that, compared with WT mice, JMJD6-deficient mice are more resistant to VSV infection with more IFN-I production and reduced viral load in livers. Our findings reveal a novel mechanism to downregulate innate immune responses mediated by RNA viral infection, which allows the host to prevent undue immune responses and sustain homeostasis. [ABSTRACT FROM AUTHOR]