In mice, the expression of the phagolysosomal protein natural-resistance associated macrophage protein 1 (Nramp1, Slc11a1) is associated with host resistance to various intracellular pathogens. Nramp1 acts as a transporter for protons, iron, and other divalent cations, and Nramp1 functionality is associated with an enhanced activity of pro-inflammatory immune pathways, including the formation of nitric oxide (NO) via transcriptional stimulation of inducible nitric oxide synthase (iNOS) expression. As iron availability also strongly influences iNOS expression, we studied the effects of Nramp1 functionality on iron homeostasis in the RAW264.7 macrophage cell line stably transfected with functional or non-functional Nramp1. We found that macrophages lacking functional Nramp1 exhibited a significantly higher iron uptake via transferrin receptor 1 and, as a consequence of this, an increased iron release which is mediated via the iron export protein ferroportin-1. RNA-bandshift experiments for determination of iron regulatory protein activity showed that, as a net effect of the altered expression of iron transporters, the overall cellular iron content was lower in macrophages bearing functional Nramp1. Since low intracellular iron availability enhances iNOS transcription, Nramp1 could exert its effect on NO formation and other pro-inflammatory immune pathways via modulation of iron homeostasis.