CD4+CD25+Foxp3+ regulatory T (Treg) cells are crucial for maintaining immune homeostasis and controlling inflammatory diseases and are differentiated from CD4+ T cells in the thymus and the periphery. Small molecules that enhance Treg cell differentiation would be beneficial for preventing and treating chronic inflammatory and autoimmune diseases. Here, we found that anti-malarial and anti-arthritic amodiaquine (AQ) affected Treg cell development. Despite the anti-proliferative activity of AQ, AQ only moderately decreased Treg cell proliferation but substantially increased IL-2 production by Treg cells. Interestingly, AQ dose-dependently increased the expression of CD25, an IL-2 receptor α subunit, and significantly facilitated Treg cell development and significantly upregulated Treg cell markers including Foxp3 and CTLA4. Furthermore, CD25 expression was increased at early stages of Treg cell development and decreased at later stanges; however, the enhanced expression of CD25 was sustained in the presence of AQ even in the presence of blokcers of IL-2 receptor signaling. AQ directly increased CD25 gene transcription by enhancing the DNA-binding activity and transcriptional activity of nuclear receptor 4A, as evidenced by chromatin immunoprecipitation and reporter gene assay. Most critically, administration of animal model with AQ suppressed development of inflammatory colitis induced by dextran sulfate sodium or inflammatory T cells, and receiprocally regulated Treg cells and inflammatory effector cells. These results suggest that anti-malarial AQ potentiates Treg cell development through activation of nuclear receptor 4A and thus may protect against the development of inflammatory and autoimmune diseases.