Objective To investigate the effect of miR-373-3p in diabetic retinopathy (DR), as well as the underlying mechanisms. Methods Serum samples from 35 DR patients and 35 non-DR patients visiting Tianjin Fifth Central Hospital from February 2021 to February 2022 were collected, and expression levels of miR-373-3p and vascular endothelial growth factor A (VEGFA) mRNA were detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR). An in vitro DR model was constructed using high glucose (HG)-treated human retinal microvascular endothelial cells (HRMEC). HRMECs were divided into control group (5 mmol/L glucose and 25 mmol/L mannitol treatment), HG group (30 mmol/L glucose treatment), HG+miR-373-3p mimic-negative control (miR-con) group (30 mmol/L glucose treatment after transfection with miR-con), HG+miR-373-3p mimic group (30 mmol/L glucose treatment after transfection with miR-373-3p), HG+miR-373-3p+vector group (30 mmol/L glucose treatment after co-transfection with miR-373-3p and vector), and HG+miR-373-3p+vascular endothelial growth factor A (VEGFA) group (30 mmol/L glucose treatment after co-transfection with miR-373-3p and VEGFA). The expression levels of miR-373-3p, VEGFA mRNA and protein were analyzed by qRT-PCR and Western blotting. CCK-8, immunofluorescence, Transwell assay, angiogenesis assay, and Western blotting were used to evaluate HRMEC proliferation, migration and angiogenesis abilities. The relationship between miR-373-3p and VEGFA was determined by dual luciferase reporter assay. Results Compared with non-DR patients, DR patients exhibited significantly lower expression levels of miR-373-3p (P<0.05) and higher expression levels of VEGFA mRNA (P<0.05) in serum. Compared with control group, HG group showed decreased expression of miR-373-3p (P<0.05), increased expressions of the mRNA and protein of VEGFA (P<0.05), higher cell viability, proliferation rate, proliferating cell nuclear antigen (PCNA) and Cylin D1 protein, and numbers of migrating cells and angiogenesis ability (P<0.05) in HRMECs. Compared with HG+miR-con group, HG+miR-373-3p group showed increased expression of miR-373-3p (P<0.05), decreased expressions of VEGFA (P<0.05), lower cell viability, proliferation rate, PCNA and Cylin D1 protein (P<0.05), and lower numbers of migrating cells and angiogenesis ability (P<0.05) in HRMECs. Compared with HG+miR-373-3p+vector group, HG+miR-373-3p+VEGFA group showed increased expression of VEGFA (P<0.05), higher cell viability, proliferation rate, PCNA and Cylin D1 protein, and numbers of migrating cells and angiogenesis ability (P<0.05) in HRMECs. The results of dual luciferase reporter assay showed decreased enzymatic activity of luciferase after cotransfection of miR-373-3p and VEGFA sequence (P<0.05). Conclusion MiR-373-3p is lowly expressed in the serum of DR patients, and its potential mechanism may involve targeting VEGFA to inhibit HG-induced HRMEC dysfunction.