The heme protein indoleamine 2, 3-dioxygenase (IDO) is induced by the pro-inflammatory cytokine interferon-γ (IFNγ) and plays an important role in the immune response by catalyzing the oxidative degradation of L-tryptophan (L-Trp) that contributes to immune suppression and tolerance. Here we examined the mechanism by which nitric oxide (NO) inhibits human IDO activity. Exposure of IFNγ-stimulated human monocyte-derived macrophages (MDM) to NO donors had no material impact on IDO mRNA or protein expression, yet exposure of MDM or transfected Cos-7 cells expressing active IDO to NO donors resulted in reversible inhibition of IDO activity. NO also inhibited the activity of purified recombinant human IDO (rhIDO) in a reversible manner and this correlated with NO binding to the heme of rhIDO. Optical absorption and resonance Raman spectroscopy identified NO-inactivated rhIDO as a ferrous iron (Fe^II)-NO-Trp adduct. Stopped-flow kinetic studies revealed that NO reacted most rapidly with Fe^II rhIDO in the presence of L-Trp. These findings demonstrate that NO inhibits rhIDO activity reversibly by binding to the active site heme to trap the enzyme as an inactive nitrosyl-Fe^II enzyme adduct with L-Trp bound and O₂ displaced. Reversible inhibition by NO may represent an important mechanism controlling the immune regulatory actions of IDO.