Mitochondria are now considered as one major target for many therapeutic approaches. In addition, the link between sirtuin activity and mitochondrial biology has recently emerged as an important field. Studies have demonstrated that sirtuins show anti-aging, anti-inflammatory, anti-tumor effects and so on, however, SIRT1 has also been reported to play an important role in the pathogenesis of various inflammatory disorders including bronchial asthma. To date, there are little experimental data on the relationship between SIRT1 and mitochondrial ROS in respiratory disorders. In this study, we have found that the SIRT1 expression and activity was significantly increased in LPS-induced lung inflammation with showing the increased generation of total cellular ROS and mitochondrial ROS, the increased nuclear NAD levels, the increased plasma exudation, the increased numbers of airway inflammatory cells in BAL fluids, the histologic changes in lung tissues, the increased nuclear translocation of NF-κB, and the increase in the levels of inflammatory cytokines. These findings were attenuated by the administration of mitochondrial ROS inhibitor, NecroX-5 or -7. Moreover, the administration of the SIRT1 inhibitor, sirtinol significantly reduced the LPS-induced lung inflammation, pathologic damage, and plasma exudation. These findings suggest that the interaction between mitochondrial ROS and SIRT1 contributes to LPS-induced lung injury cooperatively, providing a new target for the therapeutic approach to various lung inflammatory disorders including infectious diseases.