The phospholipase A₂ activity of peroxiredoxin 6 is inhibited by the transition state analogue, 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol (MJ33). This activity is required for the activation of NADPH oxidase, type 2. The present study evaluated the effect of MJ33 on manifestations of acute lung injury. Mice were injected intratracheally (IT) with lipopolysaccharide from E. coli 0111:B4 (LPS, 1 or 5 mg/kg), either concurrently with LPS or 2 h later, and evaluated for lung injury 24 h later. MJ33 inhibited reactive oxygen species (ROS) generation by lungs when measured at 24 h after LPS. LPS at either low or high dose significantly increased lung infiltration with inflammatory cells, secretion of pro-inflammatory cytokines (IL-6, TNF-α) and a chemokine (MIP-2), expression of lung vascular cell adhesion molecule (VCAM), lung permeability (protein in bronchoalveolar lavage fluid, leakage of FITC-dextran, lung wet to dry weight ratio), tissue lipid peroxidation (thiobarbituric acid reactive substances, 8-isoprostanes), tissue protein oxidation (protein carbonyls), and activation of NF-B. MJ33, given either concurrently or 2 h subsequent to LPS, significantly reduced all of these measured parameters. Previous studies of toxicity showed a high margin of safety for MJ33 in the intact mouse. Thus, we have identified MJ33 as a potent, non-toxic, and specific mechanism-based inhibitor of NOX2-mediated ROS generation that protects mice against lung injury associated with inflammation.