Indoleamine 2,3-dioxygenase (IDO1) is a heme enzyme that catalyzes the oxygenation of the indole ring of tryptophan to afford N-formylkynurenine. This activity significantly suppresses the immune response, mediating inflammation and autoimmune reactions. These consequential effects are regulated through redox changes in the heme cofactor of IDO1, which autoxidizes to the inactive ferric state during turnover. This change in redox status increases the lability of the heme cofactor leading to further suppression of activity. The cell can thus regulate IDO1 activity through the supply of heme and reducing agents. We show here that polysulfides bind to inactive ferric IDO1 and reduce it to the oxygen-binding ferrous state, thus activating IDO1 to maximal turnover even at low, physiologically significant concentrations. The on-rate for hydrogen disulfide binding to ferric IDO1 was found to be >10(6) M(-1 )s(-1) at pH 7 using stopped-flow spectrometry. Fe K-edge XANES and EPR spectroscopy indicated initial formation of a low-spin ferric sulfur-bound species followed by reduction to the ferrous state. The mu M affinity of polysulfides for IDO1 implicates these polysulfides as important signaling factors in immune regulation through the kynurenine pathway. Tryptophan significantly enhanced the relatively lower-affinity binding of hydrogen sulfide to IDO1, inspiring the use of the small molecule 3-mercaptoindole (3MI), which selectively binds to and activates ferric IDO1. 3MI sustains turnover by catalytically transferring reducing equivalents from glutathione to IDO1, representing a novel strategy of upregulating innate immunosuppression for treatment of autoimmune disorders. Reactive sulfur species are thus likely unrecognized immune-mediators with potential as therapeutic agents through these interactions with IDO1.