ObjectiveLow‐density lipoprotein (LDL) is not atherogenic. Oxidized LDL (ox‐LDL) produces reactive oxidative species (ROS) and is critical to atherosclerosis. N‐acetylcysteine (NAC) has anti‐atherosclerotic effect with largely unknown mechanisms. The present study aimed to determine if NAC could attenuate LDL oxidation in vivo.Approach and ResultsMeasurable serum level of human ox‐LDL was detected 30 min after a single dose intravenous injection of human native LDL into male C57BL/6 mice, reached the peak in 3 hours, then started to decline, and became undetectable in 12 hours. NAC treatment significantly reduced serum ox‐LDL level with no detectable serum ox‐LDL 6 hours after injection. No difference in ox‐LDL clearance was observed in NAC‐treated animals. NAC treatment also significantly decreased serum ox‐LDL level in patients with coronary artery disease and hyperlipidemia with no effect on LDL level. Intracellular and extracellular ROS production was significantly increased in the mice treated with native LDL, or ox‐LDL that was completely prevented with NAC treatment.ConclusionNAC attenuated native LDL in vivo oxidation and ROS formation from ox‐LDL.Sources of fundingNIH R01 HL094650