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Objective: The present study aims to determine hydrogen sulfide (H 2 S) concentrations of the aqueous humor from patients with diabetic retinopathy (DR) to compare its levels in the anterior segments, also to investigate its effect on the retinal microvascular endothelial cells under high glucose condition., Methods: AH samples were collected from patients with proliferative diabetic retinopathy (n = 11), non-proliferative diabetic retinopathy (n = 12) and diabetic patients without DR as controls (n = 12). There were 5 patients with PDR received intraocular anti-VEGF injection (Lucentis). Cultured RF/6A cells were grouped into control group, mannitol group, high glucose group and NaHS co-administrated high glucose group. Concentrations of H 2 S were detected by chemical assay. Cell apoptosis was evaluated by flow cytometry., Results: A significantly higher H 2 S level was observed in AH samples of PDR patients among other groups. The H 2 S level of DR group was higher than that of control group. Decreased H 2 S levels in the AH of post-injected PDR patients were observed compared with their AH samples before the anti-VEGF injection. In cell culture, low concentration of NaHS can reverse high-glucose-induced apoptosis of RF/6A cells., Conclusion: Our study revealed increased H 2 S levels in the anterior segments of different DR patients. The anti-VEGF injection reduced the H 2 S level in AH from PDR patients. The study suggested that H 2 S may serve as a biomarker in the progression of PDR. On the other hand, the H 2 S donor exerted a protective effect on retinal vascular endothelial cells against high-glucose-induced apoptosis.