Background miR-450a-5p was involved in fat formation, but its role in insulin resistance remains unclear. This study further investigated the effects of miR-450a-5p in endothelial cells, with the aim of finding a potential target for diabetes mellitus. Methods Human umbilical vein endothelial cells (HUVECs) were severally treated with low-glucose, high-glucose, methylglyoxal (MGO), and insulin only or plus MGO. miR-450a-5p was up-regulated or down-regulated in treated HUVECs. miR-450a-5p expression in cells was measured by quantitative real-time polymerase chain reaction (qRT-PCR) assays. The cell activity was determined through MTT experiments. Transwell assay and oil red O staining were used for the detection of cell invasion and fat formation. The expressions of eNOS/AKT pathway-related proteins in cells were assessed by Western blot (WB) analysis. Furthermore, the target gene of miR-450a-5p was analyzed by double-luciferase reporter analysis, and its influence in eNOS/AKT pathway was estimated. Results miR-450a-5p decreased obviously in endothelial cells with high-glucose and MGO. Through in vitro cell experiments, we knew that MGO could not only intensify the activity of endothelial cells, but also accelerate cell invasion and fat accumulation, which could be reversed by up-regulated miR-450a-5p. Moreover, MGO inhibited eNOS/AKT pathway activation and NO release mediated by insulin, which were eliminated by up-regulated miR-450a-5p. Furthermore, CREB was the target gene of miR-450a-5p that had an activation effect on the eNOS/AKT pathway. Conclusions Up-regulated miR-450a-5p eliminated MGO-induced insulin resistance via targeting CREB, which might be a potential target to improve insulin resistance and benefit patients with related diseases.