Although sirtuin 1 (Sirt1) has been found to be involved in diabetic vasculopathy and high glucose (HG)‑mediated endothelial injury, the underlying mechanisms remain to be fully elucidated. The aim of the present study was to investigate the role of Sirt1 in HG‑induced endothelial injury and its potential mechanism. In the present study, it was demonstrated that HG triggers the downregulation of Sirt1 by activating microRNA‑195 in human umbilical vein endothelial cells (HUVECs), as determined by western blot analysis in vivo and in vitro. Furthermore, a lower expression of Sirt1 was correlated with glucose metabolic abnormalities, aortic endothelial dysfunction and endothelial apoptosis as evidenced by western blot analysis and ELISA in mice. By contrast, the loss of Sirt1 evoked mitochondrial fission factor (Mff)‑mediated mitochondrial fission through the c‑Jun N‑terminal kinase (JNK) pathway, which contributes to the apoptosis of HUVECs. In addition, Sirt1 deficiency downregulated the migration of HUVECs through F‑actin dyshomeostasis. Collectively, the results identify Sirt1 as a protective factor, which inhibits the JNK/Mff/mitochondrial fission pathway and sustains F‑actin homeostasis, and has potential implications for novel approaches to diabetic vasculopathy.