Iron homeostasis is mainly controlled by the liver-produced hepcidin peptide, which induces the degradation of the ferroportin iron exporter and thus regulates serum iron level. Hepcidin transcription is clearly up-regulated by the pro-inflammatory cytokine IL-6 and down-regulated, in the case of iron depletion, at least via HIF transcription factors. In addition, in vivo iron overload up-regulates hepcidin, but this cannot be reproduced in cell culture or isolated hepatocytes. Here, we investigated the steady state mRNA levels of a series of genes involved in iron metabolism in hepatic HepG2, intestinal Caco-2, and monocyte/macrophage THP-1 cell lines under different iron and culture conditions. Our results showed that iron-saturated transferrin up-regulated hepcidin mRNA synthesis from HepG2 via cross-talk with macrophages or enterocyte cytokine-producing cells, whereas non-transferrin-bound iron down-regulated hepcidin, likely due to missing TfR-iron-transferrin uptake.