Forkhead box P3 (Foxp3) is the major transcription factor controlling the development and function of regulatory T (Treg) cells. Previous studies have indicated epigenetic regulation of Foxp3 expression. Here, we investigated whether the deoxyribonucleic acid (DNA) methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza) applied peripherally could modulate central nervous system (CNS) inflammation, by using a mouse experimental autoimmune encephalomyelitis (EAE) model. We found that disease activity was inhibited in a myelin oligodendrocyte glycoprotein (MOG) peptide-induced EAE mouse briefly pretreated with low-dose (0.15 mg/kg) 5-Aza, ameliorating significant CNS inflammatory responses, as indicated by greatly decreased proinflammatory cytokines. On the contrary, control EAE mice expressed high levels of IFN-γ and interleukin (IL)-17. In addition, 5-Aza treatment in vitro increased GFP expression in CD4(+)GFP(-) T cells isolated from GFP knock-in Foxp3 transgenic mice. Importantly, 5-Aza treatment increased Treg cell numbers, in EAE mice, at both disease onset and peak. However, Treg inhibition assays showed 5-Aza treatment did not enhance per-cell Treg inhibitory function, but did maintain a lower activation threshold for effector cells in EAE mice. In conclusion, 5-Aza treatment prevented EAE development and suppressed CNS inflammation, by increasing the number of Treg cells and inhibiting effector cells in the periphery.