Background: β2-Microglobulin (β2MG) and carbonyl stress are reported to contribute to the development of dialysis-related amyloidosis. The aim of this study was to determine whether the purity of dialysate affects plasma levels of β2MG and pentosidine (a surrogate marker of carbonyl stress) in hemodialysis patients. Methods: Sixteen patients on hemodialysis with a polysulfone membrane participated in this study. We switched the dialysate from conventional dialysate (endotoxin level 0.055–0.066 endotoxin units (EU)/ml) to ultrapure dialysate (endotoxin level <0.001 EU/ml), followed patients for 6 months, and then switched back to conventional dialysate once again. Plasma levels of β2MG, pentosidine, CRP and interleukin-6 (IL-6) were determined before the switch to ultrapure dialysate, 1 and 6 months after the switch to ultrapure dialysate, and 1 month after the switch back to conventional dialysate. Results: The switch from conventional to ultrapure dialysate significantly decreased plasma levels of β2MG, from 30.1 ± 1.4 to 27.1 ± 1.4 mg/dl (p < 0.05) and pentosidine, from 1,535.8 ± 107.5 to 1,267.6 ± 102.9 nmol/l (p < 0.01) after 1 month of use. The change of dialysate also significantly decreased plasma levels of CRP, from 0.28 ± 0.09 to 0.14 ± 0.05 mg/dl (p < 0.05) and IL-6, from 9.4 ± 2.7 to 3.5 ± 0.8 pg/ml (p < 0.01) over the 1-month period. These changes in plasma levels of β2MG, pentosidine, CRP and IL-6 were maintained over 6 months after switching to ultrapure dialysate and returned to basal levels by switching back to a conventional dialysate. Conclusions: Ultrapure dialysate decreases plasma levels of β2MG, pentosidine and inflammatory markers in hemodialysis patients. The use of ultrapure dialysate might be useful in preventing and/or treating complications of dialysis, such as dialysis-related amyloidosis, atherosclerosis and malnutrition.