Multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) are neuroinflammatory conditions where inflammatory CD4 T cells play a major role. Forkhead box P3 (Foxp3) regulatory T (Treg) cells suppress inflammation and an increase in their numbers and activity is beneficial for MS and EAE. However, studies have shown that Treg cells can transdifferentiate to pathogenic Th17 cells under inflammatory conditions. Drugs that stimulate Treg cell induction and their resistance to inflammatory stimuli are necessary to develop effective therapies to treat MS. Here, we show that primaquine (PQ), an anti-malarial drug, suppresses EAE through the stimulation of Foxp3 Treg cells. PQ-elicited Treg cells are refractory to inflammatory stimuli and suppress EAE. Additionally, PQ-elicited Foxp3 Treg cells were more efficient in suppressing the proliferation of responder cells compared to PBS-elicited Treg cells. Although PQ does not directly induce Foxp3 Treg cell differentiation from naïve T cells, it modulated dendritic cells (DCs) to induce Foxp3 Treg cells in an indoleamine 2,3 dioxygenase (IDO)-dependent manner. Together, our results show that PQ elicits Foxp3 Treg cells with a superior suppressive activity to reduce EAE. PQ has the potential as a safe and effective treatment for MS and other CNS autoimmune inflammatory diseases.