Background: Recent investigation has shown that on-line hemodiafiltration (HDF) can reduce the amount of recombinant human erythropoietin (rhEPO) deemed necessary to reach the target hematocrit. The aim of this study was to analyze the potential effect of on-line HDF on rhEPO resistance in relation to iron utilization and anemia-related parameters, when compared to conventional hemodialysis (HD). Methods: Ninety-two chronic uremic patients were treated with conventional HD and then shifted to on-line HDF. Measurements of various erythropoiesis-related parameters were collected during HD and on-line HDF periods for statistical analysis for erythropoietin resistance. Results: Patients treated with on-line HDF switching from conventional HD significantly contributed to the reduction of EPO dose to reach a higher mean hematocrit level (31.8 ± 4.4% vs. 29.5 ± 3.9%, p < 0.001) and a reduction of the serum ferritin level (322.5 ± 268.4 vs. 544.9 ± 642.4, p < 0.001). The median EPO/Hct ratio was greater in the HD period (504.6 ± 310.1) than in the on-line HDF period (307.6 ± 334.4) (p < 0.001). These results indicated a reduced EPO resistance and improved iron utilization by on-line HDF. By multiple regression analysis, the significant predictors of EPO resistance are ferritin, transferrin, albumin, and TACurea (Time average concentration of urea) in HD treatment. In on-line HDF modality, in addition to ferritin and albumin, the duration of on-line HDF is a negative predictor in EPO resistance. Conclusion: When on-line HDF is recommended to chronic dialysis patients, long-term use of this technique provides an efficient means of achieving the goal of an elevated hemoglobulin by reducing EPO resistance, improved iron utilization and may further improve the quality of life.