Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiates innate antiviral immune response. How the binding of viral RNA to and activation of the RLRs are regulated remains enigmatic. In this study, we identified ZCCHC3 as a positive regulator of the RLRs including RIG-I and MDA5. ZCCHC3 deficiency markedly inhibited RNA virus-triggered induction of downstream antiviral genes, and ZCCHC3-deficient mice were more susceptible to RNA virus infection. ZCCHC3 was associated with RIG-I and MDA5 and functions in two distinct processes for regulation of RIG-I and MDA5 activities. ZCCHC3 bound to dsRNA and enhanced the binding of RIG-I and MDA5 to dsRNA. ZCCHC3 also recruited the E3 ubiquitin ligase TRIM25 to the RIG-I and MDA5 complexes to facilitate its K63-linked polyubiquitination and activation. Thus, ZCCHC3 is a co-receptor for RIG-I and MDA5, which is critical for RLR-mediated innate immune response to RNA virus. •ZCCHC3 mediates viral RNA-triggered innate immune response•ZCCHC3 binds to viral RNA•ZCCHC3 acts as a co-receptor for RIG-I and MDA5•ZCCHC3 mediates polyubiquitination and activation of RIG-I and MDA5 by TRIM25 Recognition of viral RNA by RIG-I-like receptors (RLRs) initiates innate antiviral response. Lian et al. demonstrate that ZCCHC3 is a co-receptor for RLRs, thereby acting as an important modulator of innate antiviral response.