Infection of dendritic and glial cells with Theiler's murine encephalomyelitis virus (TMEV) induces various cytokines via Tolllike receptor- and melanoma differentiation-associated gene 5 (MDA5)-dependent pathways. However, the involvement and role of MDA5 in cytokine gene activation and the pathogenesis of TMEV-induced demyelinating disease are largely unknown. In this study, we demonstrate that MDA5 plays a critical role in the production of TMEV-induced alpha interferon (IFN-α) during early viral infection and in protection against the development of virus-induced demyelinating disease. Our results indicate that MDA5-deficient 129SvJ mice display significantly higher viral loads and apparent demyelinating lesions in the central nerve system (CNS) accompanied by clinical symptoms compared with wild-type 129SvJ mice. During acute viral infection, MDA5-deficient mice produced elevated levels of chemokines, consistent with increased cellular infiltration, but reduced levels of IFN-α, known to control T cell responses and cellular infiltration. Additional studies with isolated CNS glial cells from these mice suggest that cells from MDA5-deficient mice are severely compromised in the production of IFN-α upon viral infection, which results in increased cellular infiltration and viral loads in the CNS. Despite inadequate stimulation, the overall T cell responses to the viral determinants were significantly elevated in MDA5-deficient mice, reflecting the increased cellular infiltration. Therefore, the lack of MDA5-mediated IFN-α production may facilitate a massive viral load and elevated cellular infiltration in the CNS during early viral infection, leading to the pathogenesis of demyelinating disease.