© 2020 Elsevier B.V. and Mitochondria Research Society Diabetic retinopathy is thought to be trigger by glucose- induced oxidative stress which leads to an increase of the mitochondrial permeability through opening the permeability transition pore (MTP). In several cell types, hexokinases interact with the mitochondria regulating MTP opening, avoiding cytochrome c release. We studied HK I mitochondrial proportion in control and streptozotocin-induced diabetic rat retinas. In the normal retina, 50% of HK I was linked to mitochondria, proportion that did not change up to 60 days of diabetes. Mitochondria from normal and diabetic rat retinas showed a limited swelling, and similar cytochrome c levels. G-6-P and glycogen content increased 3–6-fold in diabetic rat retinas, while lactate content did not vary. Results suggest that mitochondrial bound HK produce G-6-P and drove it to glycogen synthesis, controlling ROS production and lactate toxicity.