Hepcidin is an antimicrobial peptide involved in regulating iron homeostasis. It is induced by iron overload and decreased by hypoxia and anemia. Hepcidin regulates iron metabolism by inhibiting iron absorption by the duodenum and by inhibiting macrophage iron recycling. Hepcidin is induced in hepatocytes during the acute-phase response by IL-6. Previously, we have shown that hepcidin is not induced in macrophages by IL-6 but is induced by the synergistic interaction of IFN-gamma and Mycobacterium tuberculosis infection. In the present study, we examined the pathways involved in inducing macrophage hepcidin expression. We show that TLRs TLR2 and TLR4 and the transcription factor STAT1 are required for induction of hepcidin mRNA. Hepcidin promoter activity is also synergistically induced in RAW264.7 macrophages by IFN-gamma and M. tuberculosis. NF-kappaB and C/CEBP binding sites are required for promoter activity. Binding of NF-kappaB (p50/p65) to the NF-kappaB site and STAT1 and C/EBPbeta to the C/CEBP site was confirmed by EMSA. Knockdown of STAT1 and C/EBPbeta expression in RAW264.7 cells with siRNA plasmids inhibited hepcidin promoter activity induced by IFN-gamma and M. tuberculosis. Together, these studies demonstrate that macrophage hepcidin expression is induced by the activation of STAT1 and NF-kappaB and the induction of C/EBPbeta expression.