Retinal homeostasis is under both diurnal and circadian regulation. We sought to investigate the diurnal expression of autophagy proteins in normal rodent retina and to determine if this is impaired in diabetic retinopathy. C57BL/6J mice and Bio-Breeding Zucker (BBZ) rats were maintained under a 12h/12h light/dark cycle and eyes, enucleated over a 24 h period. Eyes were also collected from diabetic mice with two or nine-months duration of type 1 diabetes (T1D) and Bio-Breeding Zucker diabetic rat (BBZDR/wor rats with 4-months duration of type 2 diabetes (T2D). Immunohistochemistry was performed for the autophagy proteins Atg7, Atg9, LC3 and Beclin1. These autophagy proteins (Atgs) were abundantly expressed in neural retina and endothelial cells in both mice and rats. A differential staining pattern was observed across the retinas which demonstrated a distinctive diurnal rhythmicity. All Atgs showed localization to retinal blood vessels with Atg7 being the most highly expressed. Analysis of the immunostaining demonstrated distinctive diurnal rhythmicity, of which Atg9 and LC3 shared a biphasic expression cycle with the highest level at 8:15 am and 8:15 pm. In contrast, Beclin1 revealed a 24-h cycle with the highest level observed at midnight. Atg7 was also on a 24-h cycle with peak expression at 8:15am, coinciding with the first peak expression of Atg9 and LC3. In diabetic animals, there was a dramatic reduction in all four Atgs and the distinctive diurnal rhythmicity of these autophagy proteins was significantly impaired and phase shifted in both T1D and T2D animals. Restoration of diurnal rhythmicity and facilitation of autophagy protein expression may provide new treatment strategies for diabetic retinopathy.