Experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis, is triggered by myelin-specific T effector cells primed in peripheral lymph nodes that migrate to the central nervous system (CNS) where they are reactivated and induce demyelination. First-line treatment for disease relapses is a high dose of glucocorticoids for five consecutive days. Although melatonin ameliorates the severity of EAE by modulating T-cell responses, its action mechanism remains unclear. Our objective was to study the effects of melatonin on the peripheral priming of T cells, their migration, and reactivation to the CNS, as well as to evaluate combined treatment with melatonin and methylprednisolone on neuroinflammation.EAE mice were used to analyze the frequency and/or expression of co-stimulatory/co-inhibitory molecules, adhesion molecules, and chemokine ligands/receptors in immune populations, both in the lymph nodes and in the CNS, in addition to analyzing the integrity of the blood-brain barrier (BBB). Additionally, the effector/regulatory profile of the infiltrating populations was quantified after the combined treatment.Melatonin suppressed T cell priming reducing their activation and migration. It also decreased immune infiltration, preserving BBB integrity, and down-regulating chemokines and adhesion molecules. In the CNS, melatonin reduced the expression of co-stimulatory/co-inhibitory molecules in microglia, macrophages, and CD4+ cells, and increased T cell-mediated suppressive responses.In conclusion, melatonin regulates key pathological steps in EAE, both in the periphery and in the CNS, pointing to the possible use of this molecule in the clinical context of MS, both as monotherapy or adjuvant therapy.