Aim Elevated plasma homocysteine impairs vasomotor regulation. Hyperhomocysteinemia (HH) is an independent risk factor for atherosclerosis, which causes peripheral arterial occlusive disease. Thus, we examined whether HH induced by oral methionine load modulated in vivo angiogenesis in a rat model of hindlimb ischemia. Methods Male Sprague-Dawley rats were divided into 2 groups: the control group was given tap water, and the other group was made HH by daily intake of L-methionine (1 g/kg body weight) in drinking water. At the 14th day of the dietary modification, the left femoral artery and vein were excised, and the extents of angiogenesis and collateral vessel formation in the ischemic hindlimb were examined for subsequent 4 weeks. Results Plasma homocysteine levels significantly increased, and plasma levels of nitric oxide and tissue contents of nitric oxide and cyclic guanosine 3, 5-monophosphate significantly decreased in the HH group. Serial laser Doppler blood perfusion analysis revealed significant decrease in the ratio of ischemic/normal hindlimb blood perfusion at postoperative days 7,14,21 and 28. Postmortem angiography of the ischemic limb revealed a significant decrease in the angiographic score at postoperative day 14 in the HH group. Finally, immunohistochemical examination of the ischemic tissue sections showed a significant decrease in capillary density in the HH group. Conclusions In summary, oral methionine-induced HH impairs ischemia-induced angiogenesis and collateral vessel formation in a rat model of chronic hindlimb ischemia. The mechanism appears to be mediated at least in part by decreased bioavailability of endothelium-derived nitric oxide (EDNO) in the HH state.