The elongation factor Tu GTP binding domain-containing protein 2 (EFTUD2) was identified as an anti-hepatitis C virus (HCV) host factor in our recent genome-wide small interfering RNA (siRNA) screen. In this study, we sought to further determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2 and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and JAK-STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver tissue from in HCV-infected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. EFTUD2 inhibited HCV infection by inducing expression of the interferon (IFN)-stimulated genes (ISGs) in Huh7 cells. However, its impact on HCV infection was absent in both RIG-I knockdown Huh7 cells and RIG-I-defective Huh7.5.1 cells, indicating that the antiviral effect of EFTUD2 is dependent on RIG-I. Furthermore, EFTUD2 upregulated the expression of the RIG-I-like receptors (RLRs) RIG-I and MDA5 to enhance the innate immune response by gene splicing. Functional experiments revealed that EFTUD2-induced expression of ISGs was mediated through interaction of the EFTUD2 downstream regulators RIG-I, MDA5, TBK1, and IRF3. Interestingly, the EFTUD2-induced antiviral effect was independent of the classical IFN-induced JAK-STAT pathway. Our data demonstrate that EFTUD2 restricts HCV infection mainly through an RIG-I/MDA5-mediated, JAK-STAT-independent pathway, thereby revealing the participation of EFTUD2 as a novel innate immune regulator and suggesting a potentially targetable antiviral pathway. IMPORTANCE Innate immunity is the first line defense against HCV and determines the outcome of HCV infection. Based on a recent highthroughput whole-genome siRNA library screen revealing a network of host factors mediating antiviral effects against HCV, we identified EFTUD2 as a novel innate immune regulator against HCV in the infectious HCV cell culture model and confirmed that its expression in HCV-infected liver tissue is inversely related to HCV infection. Furthermore, we determined that EFTUD2 exerts its antiviral activity mainly through governing its downstream regulators RIG-I and MDA5 by gene splicing to activate IRF3 and induce classical ISG expression independent of the JAT-STAT signaling pathway. This study broadens our understanding of the HCV innate immune response and provides a possible new antiviral strategy targeting this novel regulator of the innate response.