In type 2 diabetes mellitus (T2DM), oxidative stress induces endothelial dysfunction (ED), which is closely related to the formation of atherosclerosis. However, there are few effective drugs to prevent and cure it. Citronellal (CT) is an aromatic active substance extracted from citronella plants. Recently, CT has been shown to prevent ED, but the underlying mechanism remains unclear. The purpose of this study was to investigate whether CT ameliorated T2DM-induced ED by inhibiting the TRPM2/NHE1 signal pathway. Transient receptor potential channel M2 (TRPM2) is a Ca2+-permeable cation channel activated by oxidative stress, which damages endothelial cell barrier function and further leads to ED or atherosclerosis in T2DM. The Na+/H+ exchanger 1 (NHE1), a transmembrane protein, also plays an important role in ED. Whether TRPM2 and NHE1 are involved in the mechanism of CT improving ED in T2DM still needs further study. Through the evaluations of ophthalmoscope, HE and Oil red staining, vascular function, oxidative stress level, and mitochondrial membrane potential evaluation, we observed that CT not only reduced the formation of lipid deposition but also inhibited ED and suppressed oxidative stress-induced mitochondrial damage in vasculature of T2DM rats. The expressions of NHE1 and TRPM2 was up-regulated in the carotid vessels of T2DM rats; NHE1 expression was also upregulated in endothelial cells with overexpression of TRPM2, but CT reversed the up-regulation of NHE1 in vivo and in vitro. In contrast, CT had no inhibitory effect on the expression of NHE1 in TRPM2 knockout mice. Our study show that CT suppressed the expression of NHE1 and TPRM2, alleviated oxidative stress-induced mitochondrial damage, and imposed a protective effect on ED in T2DM rats.