Tolerogenic dendritic cells (DCs) are widely studied for their possible use in the treatment of inflammatory disorders, such as autoimmune diseases. One of the obstacles for the use of this cell-based therapy is the characterization of drugs that are able to modulate DCs. We have previously shown that chloroquine (CQ), an antimalarial agent, has the ability to modulate DCs towards a tolerogenic phenotype. 1 These tolerogenic DCs are able to suppress the development of experimental autoimmune encephalomyelitis (EAE), a T cell-driven mouse model of human multiple sclerosis. In addition, several studies have proposed that nitric oxide (NO) plays a major role in the differentiation of regulatory T cells (Tregs) and the suppression of Th1/Th17 cells. 2, 3 However, little is known about the role of DC-derived NO in the modulation of inflammatory autoimmune responses. Thus, we aimed to evaluate whether NO plays a role in the tolerogenic activity of CQ-treated DCs (CQ-DCs). We found that CQ induces DC production of NO and expression of indoleamine 2, 3-dioxygenase (IDO), as well as inducible nitric oxide synthase (iNOS). In addition, CQ-DCs stimulated the differentiation of Tregs at the expense of Th1/Th17 cells. On the other hand, iNOS2/2 DCs did not acquire a tolerogenic phenotype following CQ treatment. Rather, CQ-DCsiNOS2/2 stimulated the differentiation of Th1/Th17 cells as well as Tregs. In a therapeutic approach, CQ-DCsiNOS2/2 were unable to suppress the development of EAE. Gene expression analyses of central nervous system (CNS) tissue from mice that received CQ-DCsiNOS2/2 showed an increased expression of inflammatory modulators compared with mice that received CQ-DCsWT. In this work, we show that NO is an important factor in the modulatory activity of tolerogenic dendritic cells.