--- - |2 Summary NADPH oxidase (NOX) plays an important role in inflammatory response by producing reactive oxygen species (ROS). The inhibition of NOX has been shown to induce anti‐inflammatory effects in a few experimental models. The aim of this study was to investigate the effects of diphenyleneiodonium (DPI), a NOX inhibitor, on lipopolysaccharide (LPS)‐induced acute lung injury (ALI) in a rat model. Sprague‐Dawley rats were intraperitoneally administered by DPI (5 mg/kg) 30 minutes after intratracheal instillation of LPS (3 mg/kg). After 6 hours, bronchoalveolar lavage fluid (BALF) and lung tissue were collected. The NOX activity in lung tissue was significantly increased in LPS‐treated rats. It was significantly attenuated by DPI. DPI‐treated rats showed significant reduction in the intracellular ROS, the number of inflammatory cells, and cytokines (TNF‐α and IL‐6) in BALF compared with LPS‐treated rats. In lung tissue, DPI‐treated rats showed significantly decreased malondialdehyde content and increased activity of glutathione peroxidase and superoxide dismutase compared with LPS‐treated rats. Lung injury score, myeloperoxidase activity, and inducible nitric oxide synthase expression were significantly decreased in DPI‐treated rats compared with LPS‐treated animals. Western blotting analysis demonstrated that DPI significantly suppressed LPS‐induced activation of NF‐κB and ERK1/2 and SAPK/JNK in MAPK pathway. Our results suggest that DPI may have protective effects on LPS‐induced ALI thorough anti‐oxidative and anti‐inflammatory effects which may be due to inactivation of the NF‐κB, ERK1/2, and SAPK/JNK pathway. These results suggest the therapeutic potential of DPI as an anti‐inflammatory agent in ALI. - 'Clinical and Experimental Pharmacology and Physiology, EarlyView. '