Both tolerogenicity and immunogenicity of dendritic cells (DCs) are regulated by their intracellular metabolism. As a rate-limiting enzyme of tryptophan (Trp) metabolism, indoleamine 2,3-dioxygenase (IDO) is involved in regulating the functions of numerous cell types, including DCs, a subset of which has a high capacity for producing IDO to control over-activated inflammation. To identify the mechanisms of IDO in DCs, stable DC lines with both gainand reduction-of-function of IDO were established using a recombinant DNA technique. Although the IDO variation did not affect DC survival and migration, it altered Trp metabolism and other features of DCs analyzed by high-performance liquid chromatography and flow cytometry. On the surface of the DCs, IDO inhibited co-stimulatory CD86 but promoted co-inhibitory programmed cell death ligand 1 expression, and suppressed the antigen uptake, which ultimately led to the compromised ability of DCs to activate T cells. Furthermore, IDO also suppressed IL-12 secretion but enhanced that of IL-10 in DCs, which eventually induced T cells into tolerogenic phenotypes by inhibiting the differentiation of Th1 but promoting that of regulatory T cells. Collectively, the findings of the present study demonstrated that IDO is a key molecule for tolerogenic DC induction by metabolically regulating surface molecule and cytokine expression. This conclusion may lead to the targeted development of therapeutic drugs for autoimmune diseases.