RIG-I and MDA5 are critical sensors of virus infection and detect many important human pathogens including SARS-CoV-2, influenza A virus (IAV), Zika virus and West Nile virus (WNV). MDA5 is activated by binding to double-stranded RNA generated during infection and induces strong pro-inflammatory and antiviral responses mediated largely via type I interferons. Activation of MDA5 is also implicated in monogenic (e.g. Aicardi-Goutières Syndrome) and complex polygenic (e.g. systemic lupus erythematosus) autoinflammatory diseases, demonstrating the importance of appropriate regulation of this pathway. Yet, how MDA5 is regulated is poorly defined. We employed SILAC-mass spectrometry to discover MDA5 binding partners in virus-infected cells. Our screen revealed Annexin A2 (ANXA2) as an interactor of MDA5 specifically during infection. Direct binding of ANXA2 and MDA5 was recapitulated in vitro and required Ca2+. Binding between MDA5 and ANXA2 was dependent on both the CARDs of MDA5 and the S100A10-binding domain of ANXA2. Moreover, we found that ANXA2 supports MDA5 activity by promoting its oligomerisation. By employing trancriptomic analyses, we found that loss of ANXA2 in human cells significantly blunted interferon responses to EMCV, WNV and IAV, demonstrating a role for ANXA2 in activation of both MDA5 and RIG-I. Using human induced pluripotent stem cell-derived models, we found that ANXA2 was required for potent neuronal cell responses to WNV infection, and macrophage responses to multiple viral pathogens. Thus, we find that ANXA2 is a critical mediator of the RIG-I-like receptor pathway.