Background: Hemodiafiltration (HDF) is effective in delaying the surgical need for carpal tunnel syndrome in chronic hemodialysis patients, however, predialysis β2-microglobulin levels were not reduced in most short-term studies. The aim of this study was to assess the effect of long-term and differing frequencies of on-line HDF on serum β2-microglobulin levels in comparison to high-flux hemodialysis (HD). Methods: One hundred and twelve patients in the Chang Gung Memorial Hospital Dialysis Unit were divided into three groups to receive different frequencies of on-line HDF alternating with high-flux HD. Group 1 was treated once with HDF and twice with high-flux HD per week (n = 21). Group 2 was treated twice with HDF and once with high-flux HD per week (n = 33). Group 3 was treated with HDF three times per week (n = 58). Analysis was performed to compare the serum β2-microglobulin levels in these groups and to high-flux HD. Results: After receiving HDF for a mean of 7.9 months, group 3 patients had a reduced predialysis β2-microglobulin level (22.2 ±5.3 vs. 34.8 ±6.3 mg/l, p < 0.001), postdialysis β2-microglobulin level (6.3 ± 2.0 vs. 13.8 ± 6.8 mg/l, p < 0.001) and an increased β2-microglobulin reduction rate (76.1 ± 5.6 vs. 61.1 ± 13.3%, p = 0.03) when compared to high-flux HD. A significant improvement in URR (p = 0.0004), Kt/V (p = 0.0002) and TAC urea levels (p = 0.006) but not nPCR (p = 0.122) was found after patients had been treated with on-line HDF. The β2-microglobulin reduction rate was positively correlated with the overall volume of the replacement solution per session (p < 0.0001). Patients in group 3 had lower predialysis β2-microglobulin levels than those in group 1 and group 2 (22.2 ± 5.3 vs. 25.2 ± 7.2 vs. 26.0 ± 4.2 mg/l, p = 0.02). Furthermore, an inverse correlation was found between the predialysis β2-microglobulin level and the duration of HDF, if patients were treated for more than 12 months (p = 0.031). Conclusion: On-line HDF has an increased dialysis efficiency compared to high-flux dialysis. Long-term HDF further reduced predialysis β2-microglobulin levels, thus, it may provide an improved modality for renal replacement therapy.