Background: Retinal hypoxia is linked to the onset and progression of blinding retinal vascular diseases, such as diabetic retinopathy (DR). This study aims to investigate the role and associated molecular mechanisms of HIF-1α in mice with DR. Methods: Streptozotocin (STZ) was utilized to induce diabetic mouse models. Hematoxylin and Eosin (H&E) staining and Fundus Fluorescein Angiography (FFA) assays were employed to analyze retinal structure and observe the fundus of the mice. A Western blot assay was conducted to measure the protein levels of HIF-1α, GDNF, NRTN, LC3, P16, P21, PPAR-γ, and SIRT1 in retinal tissues. PX-478, an inhibitor of HIF-1α, was administered to treat STZ-induced mice, and Evans Blue staining was performed to assess the impact of HIF-1α depletion on blood-retinal barrier (BRB) function. Results: Through H&E staining and FFA assays, we successfully established a model of diabetic retinopathy (DR) in mice. In retinal tissues isolated from STZ-induced mice, the protein levels of HIF-1α, P16, P21, and PPAR-γ were found to be elevated, while the protein levels of GDNF, NRTN, LC3, and SIRT1 were decreased. Following treatment with PX-478, the reduction in protein levels of GDNF, NRTN, LC3, and SIRT1 was halted, and the elevated protein levels of HIF-1α, P16, P21, and PPAR-γ were also suppressed. Furthermore, PX-478 demonstrated an ability to enhance the permeability of the BRB in mice with DR. Conclusions: We have demonstrated the role of HIF-1α in diabetic retinopathy (DR), which may provide a stronger theoretical basis for clinical treatment. This finding holds significant implications for preventing blindness and improving the survival rate of patients with DR.