Background: Reactive oxygen species (ROS) are generated in the process of xenobiotic detoxification and excessive ROS production is closely related to hepatotoxicity. Carbon monoxide (CO) have been reported to protect hepatocytes against oxidative stress by activation of antioxidant defence system. Objective: The purpose of this study was to prepare carbon monoxide-releasing molecule-2-loaded liposomes (CORM-2-Lip) and evaluate the hepatoprotective effects of CORM-2-Lip. Materials and Methods: CORM-2-Lip were prepared by lipid film hydration and extrusion method with phosphatidylcholine and cholesterol as bilayer matrix components at different molar ratio. The physicochemical properties of CORM-2-Lip were determined in terms of particle size, polydispersity index, zeta potential and entrapment efficiency. CO release from CORM-2-Lip was assessed by myoglobin assay. In vitro hepatoprotective effects of CORM-2-Lip were evaluated by MTT assay in tert-butyl hydroperoxide (t-BHP)-treated HepG2 cells. Results: Spherical CORM-2-Lip showed a narrow size distribution with particle size near 100 nm. The entrapment efficiency of CORM-2 in CORM-2-Lip determined by quantifying ruthenium using ICP-AES was about 40%. The sustained CO release from CORM-2-Lip was observed and the half-life of CO release increased up to 40 times compared with that of CORM-2 solution. CORM-2-Lip showed enhanced in vitro hepatoprotective effects by augmentation of cell viability up to 1.9-fold at 50 μM of CORM-2. Conclusion: From the results, CORM-2-Lip improved in vitro hepatoprotective effects of CORM-2 and demonstrated the potential as CO therapeutic vehicles for oxidative stress-mediated liver injury.