Hepcidin has recently been recognized as a hormone essential to the negative regulation of iron. Synthesis of hepcidin is increased by iron overload or inflammation, and decreased by iron deficiency, anemia and erythropoietin. Dialysis patients frequently suffer the effects of both hepcidin increasing and decreasing factors. In this study, we investigated, for the first time, pro-hepcidin in dialysis patients while minimizing or manipulating these factors. We measured the serum pro-hepcidin in 23 hemodialysis patients without inflammation (the HD group) and 10 age-matched healthy volunteers. Those patients in the HD group were assigned to an iron-deficiency group (the ID group) or a non-iron deficiency subgroup (non-ID group). The HD group was followed up for two months. Iron therapy was performed in the ID group during the follow-up period. At the end of this time we evaluated the influence of iron therapy. Pro-hepcidin was similar in the HD groups and the healthy controls (295.1 [241.9, 413.7] vs. 301.7 [280.5, 383.5] ng/mL; not significant) despite the presence of hepcidin-decreasing factors. Pro-hepcidin in the ID group was significantly lower than in the non-ID group (262.6 [233.1, 295.1] vs. 359.2 [282.3, 446.5] ng/mL; P < 0.05). In patients newly acquiring ID during follow-up without iron supplementation, pro-hepcidin fell significantly (from 444.7 [389.4, 470.1] to 299.8 [233.4, 330.4] ng/mL; P < 0.05). Pro-hepcidin also showed an increase after ID was treated with iron administration (from 246.9 [205.5, 329.1] to 344.6 [290.1, 377.9] ng/mL; not significant). Pro-hepcidin could serve as a marker for functional iron deficiency and disordered iron utilization in HD. Underlying mechanisms and improvements in measurement techniques will require additional investigation.