While dopaminergic dysfunction is believed to be a crucial role in restless legs syndrome (RLS), changes in peripheral microvasculature system such as peripheral hypoxia and altered skin temperature, have been found. This study aimed to investigate whether patients with RLS would have a cerebral and peripheral endothelial dysfunction, and this may have association with treatment responsiveness. We evaluated cerebral endothelial function using breath-holding index (BHI) on transcranial Doppler in bilateral middle cerebral artery (MCA), posterior cerebral artery (PCA) and basilar artery (BA) and peripheral endothelial function using brachial flow-mediated dilation (FMD) in 34 patients with RLS compared with age and sex-matched controls. The values of BHI in both MCA and BA were significantly lower in RLS group than control group. The values of FMD also were significantly lower in RLS patients. There was a weak correlation between BHI and FMD (p = 0.038 in Rt MCA, p = 0.032 in Lt MCA, p = 0.362 in BA) in RLS, but not in controls. BHI differed according to treatment responsiveness. (p < 0.005). Our study suggests that RLS patients have poorer cerebral and peripheral endothelial function than controls, showing an underlying mechanism of RLS and further evidence of a possible association between RLS and cardiovascular disease.