We have previously shown that human monocyte-derived dendritic cells (DC) express indoleamine 2,3-dioxygenase (IDO), as well as several other enzymes of the kynurenine pathway at the mRNA level upon maturation. The tolerogenic mechanisms of this pathway remain unclear. Here we show that LPS-treated DC metabolize tryptophan as far as quinolinate. We found that IDO contributes to LPS and TNF-α + poly(I:C)-induced DC maturation since IDO inhibition using two different inhibitors impairs DC maturation. IDO knock-down using short-hairpin RNA also led to diminished LPS-induced maturation. In line with these results, the tryptophan-derived catabolites 3-hydroxyanthranilic acid and 3-hydroxykynurenine increased maturation of LPS-treated DC. Concerning the molecular mechanisms of this effect, IDO acts as an intermediate pathway in LPS-induced production of reactive oxygen species and NF-κB activation, two processes that lead to DC maturation. Finally, we show that mature DC expand CD4+CD25high regulatory T cells in an IDO-dependent manner. In conclusion, we show that IDO constitutes an intermediate pathway in DC maturation leading to expansion of CD4+CD25high regulatory T cells.