Diabetes was an increasing public health problem nowadays. Accumulating evidences had shed a light on the involvement of endothelial cell dysfunction in the pathogenesis of diabetes-associated vascular diseases. MiR-21, a multiple-functional miRNA, was evidenced to be involved in endothelial dysfunction, however, the underlying molecular mechanisms were still unknown. In current study, we investigated the intrinsic link between miR-21 and high glucose-induced endothelial dysfunction. We demonstrated that expression of miR-21 was elevated in circulating endothelial progenitor cells from diabetes patients. Further, inhibition of miR-21 markedly enhanced high glucose-induced endothelial cytotoxicity. Furthermore, proteomic profiling was applied to analyze the downstream effectors involved in miR-21-meidated protection of endothelial cells. A total of 31 proteins were positively identified, including Annexin A2, S100A4, SOD2, Thioredoxin and DAXX. Altered expression of these proteins was validated by immunoblot. Finally, mechanistic study showed that miR-21 protected endothelial cell against high glucose-induced endothelial cytotoxicity probably by inhibiting the expression of DAXX. Our findings were considered as a significant step toward a better understanding of diabetes-associated vascular diseases.