Background: Diabetic retinopathy (DR) is a common microvascular complication of diabetes and one of the most common causes of visual impairment and blindness. However, it is not yet known how abnormal retinal cell subpopulations contribute to disease progression. Methods: In this study, we used the Gene Expression Omnibus database to construct a single-cell atlas of DR and healthy samples to explore changes in the abundance of different cell subpopulations, and identify the molecular pathways potentially involved in DR. Results: Our results showed that DR was associated with significantly reduced numbers of bipolar cells, M??ller glia, retinal pigment epithelial cells, and cone photoreceptors, but was also associated with significantly greater numbers of pericytes, rod photoreceptors, anaplastic cells, and microglia. Our results suggest that subpopulations of M??ller glia, microglia, endothelial cells, and bipolar cells in DR tissues may be involved in various oxidative stress- and inflammation-related pathways. Conclusions: In summary, we showed that M??ller glia, endothelial cells, microglia, and bipolar cells in DR tissues are involved in oxidative stress- and inflammation-related pathways, which may contribute to the progression of the disease and ultimately lead to visual impairment and blindness. This study will provide a theoretical basis for further exploring the specific mechanism of DR.