Objective: We analyzed a prospectively collected vancomycin levels in 34 ESRD patients just before and after HD between Apr 2018 and Nov 2019. HD dialyzers were randomly assigned with the design of cross-over to the ESRD patients on HD administered intravenous vancomycin. HFD was F80® (Fresinius) or Polyflux 170H® (Baxter). Methods: There were no significant differences in the dry body weight, the level of pre-HD serum creatinine, the duration of HD session, and the amount of net ultrafiltration between two groups on HD with MCD or HFD. Although unadjusted RR of vancomycin using MCD was significantly greater than that of HFD (p = 0.036), RR of vancomycin was not significantly different between two dialyzers when adjusted for the duration of HD session (240 min). Results: Although the clearance of uremic toxin with LMMW by MCD was greater than that of HFD, there was no significant difference in the adjusted RR of vancomycin for the duration of HD session between two dialyzers. Therefore, vancomycin dosing protocol for HFD could be valid in the patients with ESRD on HD using MCD. Conclusions: Objective: For the treatment of Staphylococcus aureus infection in patients with end-stage renal disease (ESRD) on hemodialysis (HD), the maintenance of the target trough level of vancomycin (molecular weight 1,448 Dalton), 15~20 mg/L, is very important. Weight based dosing (WBD) and non-WBD protocols of vancomycin were developed on the pharmacokinetic/pharmacodynamics data of the ESRD patients using high-flux dialyzer (HFD). Recently developed medium cut-off dialyzer (MCD), Theranova®, revealed superior clearance of waste product with large middle molecular weight (LMMW, ~45,000 Dalton) than HFD. The aim of this study is to investigate whether the reduction ratio (RR) of vancomycin on MCD is greater than that on HFD. Methods: We analyzed a prospectively collected vancomycin levels in 34 ESRD patients just before and after HD between Apr 2018 and Nov 2019. HD dialyzers were randomly assigned with the design of cross-over to the ESRD patients on HD administered intravenous vancomycin. HFD was F80® (Fresinius) or Polyflux 170H® (Baxter). Results: There were no significant differences in the dry body weight, the level of pre-HD serum creatinine, the duration of HD session, and the amount of net ultrafiltration between two groups on HD with MCD or HFD. Although unadjusted RR of vancomycin using MCD was significantly greater than that of HFD (p = 0.036), RR of vancomycin was not significantly different between two dialyzers when adjusted for the duration of HD session (240 min). Conclusions: Although the clearance of uremic toxin with LMMW by MCD was greater than that of HFD, there was no significant difference in the adjusted RR of vancomycin for the duration of HD session between two dialyzers. Therefore, vancomycin dosing protocol for HFD could be valid in the patients with ESRD on HD using MCD.