Single-nucleotide polymorphisms in the tumor necrosis factor, alpha-induced protein 3 gene, which encodes the ubiquitin-modifying protein A20, are linked to susceptibility to multiple sclerosis (MS), a demyelinating autoimmune disease of the central nervous system (CNS). Since it is unresolved how A20 regulates MS pathogenesis, we examined its function in a murine model of MS, namely experimental autoimmune encephalomyelitis (EAE). Deletion of A20 in neuroectodermal cells (astrocytes, neurons, and oligodendrocytes; Nestin-Cre A20^fl/fl mice) or selectively in astrocytes (GFAP-Cre A20^fl/fl mice) resulted in more severe EAE as compared to control animals. In Nestin-Cre A20^fl/fl and GFAP-Cre A20^fl/fl mice demyelination and recruitment of inflammatory leukocytes were increased as compared to A20^fl/fl control mice. Importantly, numbers of encephalitogenic CD4⁺ T cells producing interferon (IFN)-γ, interleukin (IL)-17, and granulocyte–macrophage colony-stimulating factor (GM-CSF), respectively, as well as mRNA production of IFN-γ, IL-17, tumor necrosis factor (TNF), GM-CSF, IL-6, CXCL1, CCL2, and CXCL10 were significantly increased in spinal cords of Nestin-Cre A20^fl/fl and GFAP-Cre A20^fl/fl mice, respectively. Compared to A20-sufficient astrocytes, A20-deficient astrocytes displayed stronger activation of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) in response to TNF, IL-17, and GM-CSF, and of signal transducer and activator of transcription 1 (STAT1) upon IFN-γ stimulation. Due to NF-κB and STAT1 hyperactivation, A20-deficient astrocytes produced significantly more chemokines in response to these key encephalitogenic cytokines of autoimmune CD4⁺ T cells resulting in an amplification of CD4⁺ T cell recruitment to the CNS. Thus, astrocytic A20 is an important inhibitor of autoimmune-mediated demyelination in the CNS.