Infection of various cells with Theiler's murine encephalomyelitis virus (TMEV) activates the TLR- and melanoma differentiation-associated gene 5 (MDA5)-dependent pathways, resulting in the production of IL-1β via the activation of caspase-1 upon assembly of the node-like receptor protein 3 (NLRP3) inflammasome. The role of IL-1β in the pathogenesis of TMEV-induced demyelinating disease was previously investigated. However, the signaling effects of prostaglandin E2 (PGE2) downstream of the NLRP3 inflammasome on the immune responses to viral determinants and the pathogenesis of demyelinating disease are unknown. In this study, we investigated the levels of intermediate molecules leading to PGE2 signaling and the effects of blocking PGE2 signaling on the immune response to TMEV infection, viral persistence and the development of demyelinating disease. We demonstrate here that TMEV infection activates the NLRP3 inflammasome and PGE2 signaling much more vigorously in dendritic cells (DCs) and CD11b+ cells from susceptible SJL mice than in cells from resistant B6 mice. Inhibition of virus-induced PGE2 signaling using AH23848 resulted in decreased pathogenesis of demyelinating disease and viral loads in the central nervous system (CNS). In addition, AH23848 treatment caused the elevation of protective early IFN-γ-producing CD4+ and CD8+ T cell responses. Because the levels of IFN-β were lower in AH23848-treated mice but the level of IL-6 was similar, over-production of pathogenic IFN-β was modulated and the generation of IFN-γ-producing T cell responses was enhanced by the inhibition of PGE2 signaling. These results strongly suggest that excessive activation of the NLRP3 inflammasome and downstream PGE2 signaling contribute to the pathogenesis of TMEV-induced demyelinating disease.