Hyperglycemia is implicated in retinal vascular dysfunction associated with the development of diabetic retinopathy. Although release of vasodilators from the endothelium of retinal arterioles is known to play an important role in the regulation of retinal blood flow, the impact of hyperglycemia on vasodilator reactivity of these microvessels remains unclear. Herein, using a streptozotocin‐induced type‐1 diabetes model in the pig, we examined nitric oxide (NO) synthase (NOS)‐ and cyclooxygenase (COX)‐mediated dilation of isolated retinal arterioles at 2 and 6 wk of hyperglycemia (485±64 mg/dL vs. 81±23 mg/dl in age‐matched control). In diabetic retinal arterioles, dilation to endothelial NOS‐mediated agent bradykinin but not to endothelium‐independent NO donor sodium nitroprusside or COX‐mediated agent forskolin was significantly reduced in a similar manner at 2‐ and 6‐wk hyperglycemia. Dihydroethidium staining showed markedly increased levels of superoxide in diabetic arterioles, while protein expression of NAD(P)H oxidase subunit p47phox was elevated. These findings show that acute hyperglycemia selectively impairs endothelial NOS‐mediated dilation of retinal arterioles in a diabetic pig model. Enhanced production of NAD(P)H oxidase‐derived superoxide likely contributes to the reduction of NO bioavailability. Support: EY018420 (TWH); Scott & White Research Foundation (LK).