Endothelial dysfunction is a key feature of atherosclerosis. Retinal microvascular endothelial function can be assessed using noninvasive dynamic vessel imaging techniques. Whether it is impaired in subjects with coronary artery disease (CAD) is unknown. The aim of this study was to examine the relation of retinal microvascular endothelial function with CAD. Vascular studies were performed in 197 prospectively recruited subjects divided into 2 groups: those without CAD but ≥2 cardiovascular risk factors (non-CAD controls; n = 119) and those with stable CAD (n = 78). Retinal microvascular endothelial dysfunction was assessed by measuring retinal arteriolar and venular dilatation to flicker light, a nitric oxide–dependent phenomenon, expressed as percentage increase over baseline diameter. Fingertip pulse-volume amplitude was measured to calculate reactive hyperaemia index and brachial artery flow-mediated dilatation assessed as measures of peripheral microvascular and conduit vessel endothelial function, respectively. Mean retinal arteriolar dilatation was attenuated in patients with CAD compared with non-CAD controls (1.51 ± 1.51% vs 2.37 ± 1.95%, p = 0.001). Retinal arteriolar dilatation was independently associated with CAD after adjustment for age, gender, cardiovascular risk factors, and medication use (odds ratio 1.60, 95% confidence interval 1.14 to 2.25, p = 0.007). Reactive hyperaemia index and flow-mediated dilatation were not different. In conclusion, the capacity of retinal arterioles to dilate in response to flicker light is an independent predictor of the presence of CAD and suggests that retinal microvascular endothelial dysfunction is a marker for underlying CAD.