The optimal iron level in hemodialysis (HD) patients remains unclear. The hemoglobin content of reticulocytes (CHr) is a sensitive indicator of iron used for hematopoiesis. To identify the optimal iron content for HD patients, we investigated the relation between CHr levels and iron status, as well as the levels of hepcidin, a main regulator of iron metabolism. This study enrolled 181 HD outpatients treated with recombinant human erythropoietin (rHuEPO). A sensitivity analysis, using a generalized linear regression model that included the interaction term, was applied to determine the correlations between levels of CHr and those of serum ferritin (s-ft), transferrin saturation (TSAT), and hepcidin. The greatest changes in correlation coefficients for levels of s-ft and TSAT with CHr levels indicated optimal cut-off points of 50 ng/mL (≤50 ng/mL,  = 0.47 vs >50 ng/mL,  = 0.22) and 24% (≤24%,  = 0.58 vs >24%,  = 0.08), respectively. The correlation coefficient for levels of CHr and hepcidin showed that the optimal lower and upper cut-off points were 20 ng/mL (≤20 ng/mL,  = 0.52 vs >20 ng/mL,  = -0.01) and 70 ng/mL (≤70 ng/mL,  = 0.36 vs >70 ng/mL, = -0.45), respectively. This study indicates that the amount of iron in HD patients is sufficient for hematopoiesis under conditions of low s-ft and moderate TSAT levels. High levels of hepcidin could induce negative iron metabolism in hematopoiesis. Therefore, controlling hepcidin levels to within approximately 20-70 ng/mL may prevent iron deficiency and reduced Hb synthesis, and may thus facilitate effective iron utilization in hematopoiesis.