Objective To investigate the role of mitochondrial cytochrome c on hepatocyte apoptosis in non-alcoholic fatty liver disease in rabbits and its pathogenesis. Methods Forty Japanese white rabbits were randomly assigned to control group and model group. The model group was divided into three subgroups:4-week, 6-week, and 8-week groups, with 10 rabbits in each group. The model groups were subcutaneously injected with peanut oil (1.2 mL/kg), twice a week for 4 weeks, 6 weeks or 8 weeks. The rabbits of all groups were killed at the right time. Serum samples were collected to detect the serum biochemical index levels. Liver tissue samples were taken for pathological observation using HE staining. The hepatocyte apoptosis index (AI) was measured by flow cytometry, and mitochondrial permeability transition pore (MPTP) was evaluated by ultraviolet spectrophotometry. Immunohistochemistry was employed to detect the hepatic expressions of Bcl-2, Bax, CYT C and caspase-3. Western blot was performed to detect the changes of CYT C and caspase-3 protein expressions. Results The model groups showed hepatic injury and high level of TC, TG, CRP, IL-6 and TNF-α beginning from 4 weeks. With the NAFLD process, the hepatocyte apoptosis index was significantly increased at 4-8 weeks and the MPTP was gradually increased. In the model group, hepatic Bcl-2, Bax, CYT C and caspase-3 expressions were increased steadily with the time passing. Conclusions NAFLD-induced liver damage is associated with apoptosis, and the mitochondrial cytochrome c-mediated apoptotic pathway plays a role in the occurrence of NAFLD.