The aim of this study was to measure P-selectin, E-selectin, and CD-4L levels over time in chronic hemodialysis (HD) patients. Thirty stable patients with end-stage renal failure undergoing chronic HD were included in the study. Blood samples were obtained before HD for measurement of P-selectin, E-selectin, and CD-40L. Measurements were performed at month 0 (T0), 3 (T2), 8 (T3), and 13 (T4). The levels of P-selectin, E-selectin, and CD40L were also analyzed according to the occurrence of cardiovascular disease (CVD) and to CVD-related mortality. The levels of CD40L and P-selectin changed significantly over time, decreasing at month 3 and 6 and returning at the T0 levels at month 13. Conversely, E-selectin levels did not. The levels of CD40L, P-selectin and E-selectin over time did not differ significantly between patients with age ≤ 65 or > 65 years, between patients with or without CVD, or between patients who died or who survived during the follow-up. In end-stage renal failure patients undergoing chronic HD, CD40L and P-selectin, but not E-selectin, showed a transient decrease over time, and the serum levels of these molecules were not associated with CVD or with CVD-related mortality.