Endothelial dysfunction played an important role in the progression of diabetes mellitus (DM). miR-181c has been implicated in many diseases, including DM. However, the molecular mechanisms of miR-181c regulate this process remained poorly understood. Healthy ICR mice were divided into control group (n = 10) and db/db DM group (n = 10). The expression of miR-181c and Fox01 were both investigated in diabetic db/db mice or high glucose-induced endothelial cells (MAECs and END-D). Here we found that down-regulation of miR-181c and the activation of Fox01/iNOS were observed in mice and endothelial cells. Furthermore, we verified that miR-181c directly targeted and inhibited Fox01 gene expression by targeting its 3'-UTR through luciferase reporter assay. Knockdown of Fox01 reversed the up -regulation of iNOS, nitrotyrosine and the down-regulation of p-eNOS(Ser1177)/eNOS in high glucose (30 mM)-induced MAECs cells. In addition, over-expression of miR-181c could reverse the enhanced nitration stress induced by high glucose, while this effect could be attenuated by pcDNA-Fox01 in MAECs. These results shown that miR-181c attenuated nitration stress through regulating Fox01 expression and affecting endothelial cell function, which offering a new target for the development of preventive or therapeutic agents against DM. (C) 2017 Elsevier Inc. All rights reserved.