Rationale: Mitochondria have important roles in intracellular energy generation, modulation of apoptosis, and redox-dependent intracellular signaling. Although reactive oxygen species (ROS) participate in the regulation of intracellular signaling pathways, including activation of NF-B, there is only 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-B activation in neutrophils and neutrophil dependent acute lung injury. Measurements and Main Results: Inhibition of complex I with either rotenone or the anti-hyperglycemic agent metformin was associated with increased intracellular levels of both superoxide and hydrogen peroxide, as well as inhibition of LPS-induced IB- degradation, NF-B 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 results demonstrate that mitochondrial complex I plays an important role in modulating TLR4-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.