Patients on antiepileptic drugs (AEDs) may have elevated levels of plasma total homocysteine (p-tHcy). The aim of this study was to assess the effect of B-vitamin supplementation on the levels of p-tHcy and markers of endothelial activation and lipid peroxidation. A total of 33 adult patients on AEDs were identified with either fasting (Group 1, n=23) or post methionine load (PML) (Group 2, n=10) hyperhomocysteinemia. Subjects were supplemented with B-vitamins for 30 days: folic acid 0.4 mg, pyridoxine 120 mg and riboflavin 75 mg per day. After supplementation, serum folate and pyridoxal phosphate had increased, while fasting and PML p-tHcy had decreased (P<0.0001) by 36 and 26%, respectively. Prior to supplementation, the Group 1 patients had elevated levels of P-selectin and von Willebrand factor (vWF) (P=0.05 and 0.03, respectively). After supplementation, the levels of intercellular cell adhesion molecules had decreased (P=0.01) and E-selectin decreased nonsignificantly (P=0.07). However, the levels of vascular cell adhesion molecules had increased (P<0.0001), while lipid peroxidation were unchanged. In conclusion, the combined supplementation with folic acid, pyridoxine and riboflavin reduced fasting and PML hyperhomocysteinemia in patients on AEDs. Patients with fasting hyperhomocysteinemia had elevated levels of P-selectin and vWF, which may indicate an increased risk of cardiovascular disease. Furthermore, B-vitamin supplementation influenced endothelial activation, although the clinical implication is uncertain.