In recent years, the applications of nanoparticles have received a great attention due to their industrial and biomedical applications, while their beneficial effects suffer from controversial results at clinical stages. In the current study, cytotoxicity of cerium oxide (CeNP) nanoparticles (100 nm) were evaluated using mitochondria derived from wistar rat's liver. Isolated mitochondria from rat’s liver were divided into 7 groups including group 1 as control and group 2 to 7 as treatment group with different doses of CeNP (5, 10, 50, 100, 250 and 500mg/ml, respectively), for 24,48 and 72 hours. After exposure, oxidative stress biomarkers such as total antioxidant capacity (TAC), lipid peroxidation (LPO), total thiol groups (TTG), catalase activity (CAT) and mitochondrial viability, were determined in isolated rat liver mitochondria. Results have shown that CeNPs increase TAC, TTG, CAT, LPO and viability of mitochondria in various exposure times and confirm antioxidant properties of CeNPs in mithocondria while mitochondria is a main source for the generation of reactive oxygen species (ROS).