Diabetic retinopathy (DR) is a major cause of blindness globally. Neutrophils and neutrophil extracellular traps (NETs) are believed to play a role in the development of DR. However, the specific contribution of NETs to hyperglycemia-induced vascular endothelial cell dysfunction remains unclear. In this study, we cocultured high glucose-activated neutrophils (HGNs) with human umbilical vein endothelial cells (HUVECs) to investigate the role of NETs in high glucose-induced HUVEC dysfunction. Our findings indicate that high glucose levels promote NETs formation, which can be inhibited by a toll-like receptor (TLR) 2 antagonist and a TLR4 antagonist. It was observed that reactive oxygen species production plays a role in TLR2- but not TLR4-mediated NETs formation. Additionally, HGNs were found to promote HUVEC proliferation through phagocytosis rather than NETs. We also discovered that NETs contribute to high glucose-induced HUVEC dysfunction by enhancing neutrophil-HUVEC adhesion, inhibiting HUVEC migration, and compromising the barrier function of the cells by reducing zonula occludens-1 expression. This dysfunction could be partially mitigated by TLR2 and TLR4 antagonists. In conclusion, high glucose stimulates NETs formation, leading to vascular endothelial cell damage, and TLRs may facilitate high glucose-induced endothelial dysfunction by modulating NETs formation.