Iron plays an essential role in physiology including the control of the immune function and phenotype of macrophages [1, 2]. Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme enzyme that catalyses the rate-limiting step of L-tryptophan catabolism along the kynurenine pathway. It is robustly expressed in activated macrophages and signals for immunosuppression during inflammation and cancer [3]. The incorporation of the heme-iron prosthetic group is essential for IDO1 enzyme activity. We examined the impact of intracellular iron on the regulation of IDO1 in interferon-γ (IFNγ) and lipopolysaccharide (LPS)-stimulated primary human monocyte-derived macrophages (MDMs). Iron deprivation through the addition of the iron chelators, deferiprone (DFP) or desferrioxamine (DFO), strongly inhibited IDO1 mRNA and protein expression and enzyme activity in IFN-γ- or LPS-stimulated human MDMs. In contrast, exogenous iron supplementation did not markedly affect IDO1. To verify that the inhibitory actions of the iron chelators involved their iron chelation properties, pre-incubation of DFO or DFP with ferric-iron chloride (FeCl3) prior to addition to the MDMs was shown to significantly reverse their inhibitory activity towards IDO1. Interestingly, in IFNγ- or LPS-stimulated human MDMs iron chelation did not inhibit the phosphorylation of STAT1 and NFκB, which are essential for IDO1 transcription. This indicates that iron chelators inhibit IDO1 via targeting alternative signaling events. Our data shows that IDO1 expressed in IFNγ- or LPS-stimulated MDMs is subject to control at the level of intracellular iron availability. Therefore, the emerging link between iron homeostasis and control of macrophage immune function may relate, in part, to modulation of the immune regulatory properties of IDO1.