Diabetic retinopathy (DR) is a major cause of blindness in working adults in the industrialized world. In addition to vision loss caused by macular edema and pathological angiogenesis, DR patients often exhibit neuronal dysfunction on electrophysiological testing, suggesting that there may be an independent neuronal phase of disease that precedes vascular disease. Given the tremendous metabolic requirements of the retina and photoreceptors in particular, we hypothesized that derangements in metabolic regulation may accelerate retinal dysfunction in diabetes. As such, we induced hyperglycemia with streptozotocin in mice with monoallelic Nampt deletion from rod photoreceptors, mice lacking SIRT3, and mice lacking SIRT5 and tested multiple components of retinal function with electroretinography. None of these mice exhibited accelerated retinal dysfunction after induction of hyperglycemia, consistent with normal-appearing retinal morphology in hyperglycemic Sirt3 −/− or Sirt5 −/− mice. However, mice lacking both SIRT3 and SIRT5 (Sirt3 −/− Sirt5 −/− mice) exhibited significant evidence of inner retinal dysfunction after induction of hyperglycemia compared to hyperglycemic littermate controls, although this dysfunction was not accompanied by gross morphological changes in the retina. These results suggest that SIRT3 and SIRT5 may be involved in regulating neuronal dysfunction in DR and provide a foundation for future studies investigating sirtuin-based therapies.