Background: There is a very high prevalence of subclinical vitamin K deficiency in patients requiring hemodialysis (HD), and this problem is associated with vascular calcification and arterial stiffness. Vitamin K2 (MK-7) supplementation can improve vitamin K status in HD patients. However, the benefits of vitamin K supplementation on arterial stiffness have still not been established. The present study was conducted to evaluate the efficacy of menaquinone-7 (MK-7) supplementation on arterial stiffness in chronic HD patients. Methods: This open-label multicenter randomized clinical trial was conducted in 96 HD patients who had arterial stiffness, defined by high carotid femoral pulse wave velocity (cfPWV ≥ 10 m/s). The patients were randomly assigned to receive oral MK-7 (375 mcg once daily) for 24 weeks (n= 50) or standard care (control group;n= 46). The change in cfPWV was the primary outcome. Results: Baseline parameters were comparable between the two groups. There was no significant difference in the change in cPWV at 24 weeks between the MK-7 group and standard care [−6.0% (−20.2, 2.3) vs. −6.8% (−19.0, 7.3),p= 0.24]. However, we found that MK-7 significantly decreased cPWV in patients with diabetes [−10.0% (−15.9, −0.8) vs. 3.8% (−5.8, 11.6),p= 0.008]. In addition, the MK-7 group had a lower rate of arterial stiffness progression, compared to controls (30.2% vs. 39.5%,p= 0.37), especially in diabetes patients (21.4% vs. 72.7%,p= 0.01). No serious adverse events were observed during the 24 weeks. Conclusion: Vitamin K supplements provided a beneficial impact in lowering the rate of arterial stiffness progression in chronic hemodialysis patients with diabetes. Possible benefits on cardiovascular outcomes require further investigation.