Atherosclerotic complications have a significant effect on mortality in patients undergoing hemodialysis (HD) therapy. However, anti-atherosclerotic and cardioprotective effects of on-line hemodiafiltration (HDF) remain to be elucidated. We prospectively compared the anti-atherosclerotic and cardioprotective effects in two randomly divided groups, i.e. on-line HDF group (n = 13) and conventional HD group (n = 9) for 1 year. Surrogate markers were brachial-ankle pulse wave velocity (baPWV), intima-media thickness (IMT) of carotid artery as an atherosclerosis marker, and cardiac functional surrogate markers included left ventricular mass index (LVMI), ejection fraction (EF), and LV diastolic capacity represented as E/A and deceleration time (DT). LVMI in on-line HDF patients showed significant regression after 1 year of treatment (131.9 ± 25.8 to 116.5 ± 24.7 g/m2, P = 0.03), while LVMI in HD patients did not show any significant change (148.0 ± 47.1 to 142.3 ± 35.5 g/m2). Levels of baPWV in HD patients showed a significant increase (11.4%) from basal levels, while on-line HDF groups showed no significant increase. Furthermore, HD patients showed significant worsening of LV diastolic capacity (E/A: from 0.87 ± 0.12 to 0.79 ± 0.08, P = 0.03), while it was not shown in on-line HDF patients. Ejection fraction and IMT did not show any significant change in both groups. Serum albumin, C-reactive protein, β2 microglobulin, blood pressure, and anti-hypertensive drug use did not change in both groups. On-line HDF showed a significant improvement in LVMI and prevented a significant worsening of baPWV or LV diastolic capacity compared with patients on conventional HD therapy.