Endothelial dysfunction and vascular complications induced by hyperglycemia play an important role in the pathological development of atherosclerosis in diabetes. Humanin, a 24-amino acid mitochondria-derived polypeptide, has displayed its cytoprotective effects in diverse cell types and tissues. In the current study, we aimed to characterize the effects of humanin on high glucose-induced endothelial dysfunction. Firstly, we found that humanin treatment induced the expression of Krüppel-like factor 2 (KLF2), an essential transcriptional regulator of endothelial function, at the transcriptional level in human umbilical vein endothelial cells (HUVECs). Additionally, our results indicate that humanin treatment regulated the expression of KLF2 target genes such as endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1). Evidence demonstrated that the effects of humanin on KLF2 expression was mediated by the phosphorylation of extracellular signal regulated kinase 5 (ERK5). Furthermore, humanin restored high glucose-induced reduction of KLF2 expression. We also showed that humanin significantly reduced the expression of vascular cell adhesion molecule 1 (VCAM-1) and E-selectin. Notably, humanin treatment markedly prevented high glucose-induced attachment of the monocyte THP-1 cells to HUVECs. However, knockdown of KLF2 abolished these effects. Lastly, we report that humanin treatment inhibited high glucose-induced secretion of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). These findings suggest that humanin may have therapeutic potential for the treatment of hyperglycemia-associated endothelial dysfunction.