Abstract: In this study, the combination of in vitro experiments and network pharmacology was used to explore the mechanism of paeoniflorin in treating diabetic retinopathy. An in vitro model of retinal vascular endothelial cells under high glucose environment was prepared to explore the protective effect of paeoniflorin on diabetic retinopathy. The targets of paeoniflorin and diabetic retinopathy were obtained from TCMSP, UniProt, PubChem, PharmMapper, SwisstargetPrediction, GeneCards, OMIM, DrugBank, TTD databases, and the core targets were obtained by Venn diagram. Based on the STRING database, the protein-protein interaction network and a “drug-target-disease” network were constructed using Cytoscape; The DAVID database was used to carry out GO biological process and KEGG pathway enrichment to analyze the core target, and the AutoDock software was used to carry out molecular docking verification between paeoniflorin and the core target. In vitro experimental results show that paeoniflorin can significantly inhibit the proliferation and migration ability of retinal vascular endothelial cells and reduce the length of lumen formation. The results of network pharmacology show that a total of 154 core targets for the treatment of diabetic retinopathy with paeoniflorin have been obtained, among which higher degree targets include VEGFA, IL6, etc. The GO function is enriched to 705 GO items, mainly involving cell response to drugs, the regulation of vascular endothelial cell migration, regulation of cell proliferation and other biological processes; The KEGG pathway enrichment analysis enriched a total of 137 pathways, mainly including the AGE-RAGE signaling pathway and the PI3K-Akt signaling pathway. Molecular docking results showed that paeoniflorin molecules had good binding ability to core targets VEGFA and IL6. This study suggests that paeoniflorin may play a role in the treatment of diabetic retinopathy by regulating the proliferation, migration ability and lumen formation length of retinal vascular endothelial cells through multiple targets and pathways.