Background: Hemodiafiltration (HDF) is associated with a lower incidence of neuropathy, carpal tunnel syndrome, joint pain, and partial correction of anemia. HDF with on-line-prepared substitution fluid (OL HDF), as compared with conventional hemodialysis, increases the treatment tolerance and, as compared with standard HDF, avoids storage problems and allows a higher substitution volume at low cost. Methods: Thirty-two hemodialysis patients treated by OL HDF for at least 9 months were studied. Hemoglobin, hematocrit, iron metabolism, serum albumin, dialysis dose and dry body weight were determined under a settled condition with regular hemodialysis 3 months before the transfer to OL HDF. The same parameters were analyzed 3, 6 and 9 months after the beginning of the new treatment modality. Results: During OL HDF, hemoglobin values significantly increased in patients without addition of recombinant human erythropoietin (rHuEPO): baseline vs. 6 months 11 ± 1.7 vs. 12 ± 1.8 g/dl (p < 0.01); baseline vs. 9 months 11 ± 1.7 vs. 12 ± 1.6 g/dl (p < 0.05). In patients on a maintenance dose of rhuEPO, this could be significantly reduced, while the target hemoglobin levels were maintained (10.6 ± 0.9 g/dl): baseline 99.8 ± 50.4 U/kg/week, 3rd month 76.2 ± 43 U/kg/week, 6th month 64.3 ± 37 U/kg/week, and 9th month 59.4 ± 38.6 U/kg/week (p = 0.007, p = 0.0006, and p = 0.0007, respectively, vs. baseline). Iron metabolism, dialysis dose, dry body weight and serum albumin levels did not significantly change during the follow-up period. Further, a stability of the rHuEPO supplementation was observed in 14 patients followed up for 24 months. Conclusions: OL HDF influences anemia and rHuEPO dose. It allows considerable anemia correction in patients without rHuEPO treatment, while it significantly reduces rHuEPO doses in those on rHuEPO treatment as compared with standard hemodialysis. The rHuEPO costs are consequently reduced.