The intracellular RIG-I-like receptors recognize 5′-triphosphate viral genomic RNA and initiate the production of cytokines through mitochondria adaptor VISA. The regulation of this signal pathway is largely unknown. In this study, we report that the histone methyltransferase enhancer of zeste homolog 2 (EZH2) inhibits RIG-I signal pathway in an methyltransferase-independent manner. Knockdown EZH2 expression enhances VISA-induced activation of IFN-β promoter and NF-κB signaling. Cytosolic distributed EZH2 colocalizes with VISA and binds to its caspase recruitment domain (CARD), thus blocking its association with RIG-I. During the infection of influenza A virus (IAV) strain A/WSN/33 (WSN), EZH2 translocates to RIG-I and continuously interferes the interaction between RIG-I and VISA. Both N and C termini of EZH2 interact with VISA and attenuate its downstream signaling. WSN virus infection-induced expression of TNF-α, IFN-β, and IL-8 is inhibited by EZH2 and its catalytic dead form ΔSET. EZH2 overexpression facilitates the replications of IAV strains WSN and A/Puerto Rico/8/34 influenza virus. Knockdown EZH2 expression activates infection-induced IFN-β transcription and inhibits virus replication. We further provided evidence to show that pharmacological disruption of EZH2 expression by its inhibitor 3-deazaneplanocin A activates innate immune responses and attenuates the replication of WSN virus in HeLa, MDCK, and mouse primary bone marrow-derived macrophages, but not in IFN-deficient Vero cells. Collectively, these results revealed that EZH2 binds to VISA and interferes with the interaction between VISA and RIG-I. Targeting EZH2 activates mitochondria-mediated antiviral innate immune responses, and thus represses the replication of IAV in cells.