Hepcidin is presumed to be the master regulator of systemic iron homeostasis; its gene expression is affected by inflammation, hypoxia, iron levels, the rate of erythropoiesis, and oxidative stress. Hemojuvelin (HJV), a recently identified protein expressed in muscle, heart, and liver, has been shown to be an upstream regulator of hepcidin expression by acting as a bone morphogenetic protein (BMP) co-receptor. Consequently, HJV genetic mutations lead to the severe iron overload disorder juvenile hemochromatosis. There are two HJV isoforms: a membrane-bound and a soluble form, both of which are proposed to regulate hepcidin expression in response to changes in iron levels in vitro and in vivo. However, there is no information on how HJV is regulated. In this study, we identified E-box elements in the human HJV promoter and investigated the role of upstream stimulatory factors (USF) in the regulation of HJV expression through the E-boxes. By co-transfection experiments, we demonstrated that over-expression of USF trans-activated the HJV promoter. This was confirmed with electrophoretic mobility shift assays which showed that recombinant USF1 and USF2 bound specifically to the HJV E-box. Our data suggests that USF transcription factors are important regulators of HJV expression, and strengthens the link between USF and iron metabolism.