Hepcidin is a peptide hormone and has emerged as the central molecule regulating systemic iron homeostasis. Hepcidin inhibition could be a strategy for treating anemia of chronic disease. We previously reported the discovery of DS79182026, a new inhibitor of hepcidin production, from phenotypic screening using the human hepatocyte HepG2 cell line. In this study, we utilized a combination of affinity purification-based chemical proteomics and radioactive compound binding assay, and identified several candidate proteins. Purified recombinant proteins were subjected to radioactive compound binding assays for validation, and ALK2 and ALK3 demonstrated specific binding to the compound. Since ALK2 is known to be related to hepcidin production, we focused on ALK2 and found that its knockdown decreased hepcidin expression; we also found a strong correlation (R = 0.920) between pharmacological activity and compound affinity to ALK2. These results indicate that ALK2 is the primary target protein of our new hepcidin production inhibitors. (C) 2018 Elsevier Inc. All rights reserved.