Knowledge of hepcidin regulation is foremost gained by in vitro studies. We aimed to translate this knowledge into the human in vivo situation. Therefore, we measured serum markers as transferrin saturation (TS), soluble transferrin receptor (sTfR), and C-reactive protein (CRP) in parallel with hepcidin and prohepcidin in patients with iron metabolism disorders and controls. To assess sTfR as erythropoietic activity-associated factor in hepcidin regulation, we studied its influence on hepcidin expression in HepG2 cells. Results showed that sTfR highly associates with erythropoietic activity that strongly interfered with the iron store regulation of hepcidin. HepG2 expression results display an inverse association between hepcidin and sTfR. Inflammation was strongly related to increased hepcidin levels regardless of the iron store and erythropoietic activity status. In contrast, prohepcidin failed to correlate to any other parameter. In conclusion, these studies verify that previous conclusions based on in vitro studies on hepcidin regulation are also likely to apply to human patients. This is underscored by a simple algorithm, based on parameters reflecting the main regulating pathways, that accurately predict the actual measured hepcidin levels. Future studies are needed to validate the combined utility of this predictive algorithm together with actual measured hepcidin levels in clinical diagnosis.