Rationale: Mitochondria have important roles in intracellular energy generation, modulation of apoptosis, and redox-dependent intra-cellular signaling. Although reactive oxygen species (ROS) participate in the regulation of intracellular signaling pathways, including activation of nuclear factor (NF)-κB, thereisonly limited information concerning the role of mitochondrially derived ROS in modulating cellular activation and tissue injury associated with acute inflammatory processes. Objectives: To examine involvement of the mitochondrial electron transport chain complex I on LPS-mediated NF-κB activation in neutrophils and neutrophil-dependent acute lung injury. Methods: Neutrophils incubated with rotenone or metformin were treated with bacterial lipopolysaccharide (LPS) to determine the effects of mitochondrial complex I inhibition on intracellular concentrations of reactive oxygen species, NF-κB activation, and proin-flammatory cytokine expression. Acute lung injury was produced by intratracheal injection of LPS into control, metformin, or rotenone-treated mice. Measurements and Main Results: Inhibition of complex I with either rotenoneorthe antihyperglycemic agent metformin was associated with increased intracellular levels of both superoxide and hydrogen peroxide,aswellasinhibitionofLPS-inducedIκB- αdegradation,NF-κB nuclear accumulation, and proinflammatory cytokine production. Treatment of LPS-exposed mice with rotenone or metformin resulted in inhibition of complex I in the lungs, as well as diminished severity of lung injury. Conclusions: These resultsdemonstrate that mitochondrial complexI plays an important rolein modulating Toll-like receptor 4-mediated neutrophil activation and suggest that metformin, as well as other agents that inhibit mitochondrial complex I, may be useful in the prevention or treatment of acute inflammatory processes in which activated neutrophils play a major role, such as acute lung injury.