Objective Iron deficiency, especially functional iron deficiency, is one of the most common causes of a suboptimal response to recombinant human erythropoietin (EPO) therapy in anemic hemodialysis patients, therefore iron supplementation is important for the patients. In view of current uncertainty regarding the optimum route for iron supplementation, a prospective randomized controlled study was designed to investigate this issue.Methods Tweenty-five hemodialysis patients who had a hemoglobin concentration of 60～90g/L, a serum ferritin concentration of (100～500)g/L and transferritin saturation less than 20%, were randomized into two groups with different route of iron supplementation. ① Intravenous iron group (IV group), 13 patients, administered intravenous iron sucrose 100mg per week. ② Oral iron group (Oral group), 12 patients, oral ferrous succinate 200mg tid. All patients were treated with 100～180U/kg/week of EPO subcutaneously. The hemoglobin (Hb) concentration, hematocrit (Hct) and reticulocyte (Ret) count were monitored every two weeks, and the reticulocyte hemoglobin content (CHr), percentage of hypochromic red cells (%HRC), serum ferritin (SF) and transferritin saturation (TS) every four weeks. Results The two groups were equivalent with regard to age, sex, duration of hemodialysis and body weight. Before treatment there were no differences between the groups in Hb, Hct, Ret, CHr, %HRC, SF and TS. At the end of the trial, the Hb and Hct in both groups significantly increased respectively. The response of the Hb and Hct in IV group was markedly greater than that in oral group respectively［Hb (104.4±8.1) vs (96.3 ± 5.5) g/L, P < 0.01; and Hct 31.4% ± 2.5% vs 29.1% ± 1.6%, P<0.05, respectively］. The CHr, SF and TS in IV group increased significantly ［CHr (27.1±1.2)pg vs (29.9 ± 1.5)pg, P<0.001; SF(211±71)μg/L vs (283±62)μg/L, P<0.01; and TS 13.3%±3.4% vs 20.0%±2.9%, P<0.01, respectively］, The %HRC decreased significantly (from 5.7%±3.1% to 2.6%±1.2%, P<0.001). The CHr, %HRC, SF and TS in Oral group remained constant. There was no significant difference in average dosages of EPO between the two groups［IV group (141±11) vs Oral group (136±16)U/kg/w, P>0.05］. The serum levels of folate, VitaminB12, CRP, ALT, AST, Tbil, Alb, Cr and BUN in both groups did not change significantly. There were no severe adverse events in both groups. Conclusions The results of the study suggest that, even in iron-replete patients suffering from functional iron deficiency, those supplemented with intravenous iron have an enhanced hemoglobin response to EPO with better maintenance of iron stores, compared with those patients receiving oral iron.