Background: Low concentrations of carbon monoxide (CO) protect hepatocytes against apoptosis and confers cytoprotection in several models of liver. Mitochondria are key organelles in cell death control via their membrane permeabilization and the release of pro-apoptotic factors. Results: Herein, we show that CO prevents mitochondrial membrane permeabilization (MMP) in liver isolated mitochondria. Direct and indirect approaches were used to evaluate MMP inhibition by CO: mitochondrial swelling, mitochondrial depolarization and inner membrane permeabilization. Additionally, CO increases mitochondrial reactive oxygen species (ROS) generation, and their scavenging, by beta-carotene addition, decreases CO protection, which reveals the key role of ROS. Interestingly, cytochrome c oxidase transiently responds to low concentrations of CO by decreasing its activity in the first 5 min, later on there is an increase of cytochrome c oxidase activity, which were detected up to 30 min. Conclusion: CO directly prevents mitochondrial membrane permeabilization, which might be implicated in the hepatic apoptosis inhibition by this gaseoustransmitter.