Background: Tissue ATP depletion and oxidative stress have been associated with the severe outcomes of septic shock. One of the compensatory mechanisms to alleviate the sepsis-induced mitochondrial dysfunction could be the increase in oxidative phosphorylation efficiency (ATP/O). We propose to study liver mitochondrial function and oxidative stress and the regulatory mechanism of mitochondrial oxidative phosphorylation efficiency in an animal model of sepsis. Methods: We induced sepsis in rats by cecal ligation and perforation (CLP). Six, 24, or 36 h following CLP, we measured liver mitochondrial respiration, cytochrome c oxidase activity, and membrane permeability. We determine oxidative phosphorylation efficiency, by measuring ATP synthesis related to oxygen consumption at various exogenous ADP concentrations. Finally, we measured radical oxygen species (ROS) generation by liver mitochondria and mRNA concentrations of UCP2, biogenesis factors, and cytokines at the same end points. Results: CLP rats presented hypotension, lactic acidosis, liver cytolysis, and upregulation of proinflammatory cytokines mRNA as compared to controls. Liver mitochondria showed a decrease in ATP synthesis and oxygen consumption at 24 h following CLP. A marked uncoupling of oxidative phosphorylation appeared 36 h following CLP and was associated with a decrease in cytochrome c oxidase activity and content and ATP synthase subunit β content (slip mechanism) and an increase in mitochondrial oligomycin-insensitive respiration, but no change in mitochondrial inner membrane permeability (no leak). Upregulation of UCP2 mRNA resulted in a decrease in mitochondrial ROS generation 24 h after the onset of CLP, whereas ROS over-generation associated with slip at cytochrome c oxidase observed at 36 h was concomitant with a decrease in UCP2 mRNA expression. Conclusions: Despite a compensatory increase in mitochondrial biogenesis factors, liver mitochondrial functions remain altered after CLP. This suggests that the functional compensatory mechanisms reported in the present study (slip at cytochrome c oxidase and biogenesis factors) were not strong enough to increase oxidative phosphorylation efficiency and failed to limit liver mitochondrial ROS over-generation. These data suggest that treatments based on cytochrome c infusion could have a role in mitochondrial dysfunction and/or ROS generation associated with sepsis.