Technical Abstract: Hepcidin is the principal iron-regulatory hormone and pathogenic factor in anemia of inflammation. Patients with multiple myeloma (MM) frequently present with anemia. We showed that MM patients had increased serum hepcidin, which inversely correlated with hemoglobin, suggesting that hepcidin contributes to MM-related anemia. Searching for hepcidin-inducing cytokines in MM, we focused on BMPs and IL-6, the major transcriptional regulators of hepcidin. We developed an in vitro reporter system consisting of JuH7 cells, and human hepcidin promoter-luciferase constructs to quantify hepcidin promoter activity in response to MM patient sera. The effects of IL-6 or BMPs were selectively disrupted by mutating the promoter at STAT3-binding site (STAT3-B@) or two BMP-responsive elements (BREs), respectively. MM patient sera activated the hepcidin promoter significantly more than normal sera. Mutations in BREs abrogated the activation dramatically, while mutations in STAT3-BS had only a minor effect. Co-treatment of MM sera with anti-BMP-2/-4 or noggin-Fc blocked the induction, whereas, anti-IL-6, anti-BMP -4, -6, or -9 antibodies did not. BMP-2 immunodepleted MM sera had decreased promoter-stimulatory capacity, and ELISA assay showed that MM sera contained significantly higher BMP-2 concentrations than normal sera. Our results demonstrate that BMP-2 is a major mediator of the hepcidin-stimulatory activity of MM patient sera.