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Minimally modified low-density lipoprotein (mmLDL) is a well-known risk factor for cardiovascular diseases. The present study was designed to investigate the role of mmLDL in the endothelium-dependent relaxation of mouse mesenteric arteries. A sensitive myograph system was employed to examine the endothelial function of mesenteric arteries. mRNA and protein expression levels were determined using real-time PCR and Western blotting, respectively. The ultramicrostructure of mesenteric vascular beds was investigated using a transmission electron microscope. The results showed that mmLDL significantly impaired the acetylcholine-induced (3 × 10-10 to 1 × 10-4M) endothelium-dependent relaxation of mouse mesenteric arteries with markedly reduced pIC50 (p < 0.05) and Rmax values (p < 0.001). In addition, mmLDL increased the levels of superoxide production and nitrotyrosine concentration and impaired the endothelial microstructure with decreased KCa3.1 and KCa2.3 expression. In conclusion, mmLDL increases superoxide and nitrotyrosine levels, damages endothelial microstructure with decreased KCa3.1 and KCa2.3 expression, and ultimately attenuates relaxation mediated by nitric oxide- and endothelium-derived hyperpolarizing factor.