Objective: Obesity and insulin resistance are considered the main causes of non-alcoholic fatty liver disease (NAFLD), and oxidative stress accelerates the progression of NAFLD. Among the ROS produced, Hydrogen peroxide (H2O2) produced endogenously can cause mitochondrial dysfunction and metabolic complications in various cell types by inducing oxidative stress. H2O2 is mainly produced in peroxisomes and decomposed by catalase. In the liver, oxidative and endoplasmic reticulum (ER) stress affects the development of non-alcoholic fatty liver disease (NAFLD). Although a link between both stresses and fatty liver diseases has been suggested, few studies have investigated the involvement of catalase in fatty liver pathogenesis. Here, we show that ablation of catalase promotes fatty liver by oxidative stress. Methods: We analyzed liver extracted from 5-, 10- and 37-week-old WT and CKO mice. Next, seven-week-old WT and CKO mice were randomly divided into two groups, a normal diet (ND, n = 10) and high fat diet (HFD, 60% of kcal from fat, n = 10) for 4weeks. All values are expressed as mean ± SEM. Results: As a result, there were no morphological differences in the liver between in WT and CKO mice aged 5 and 10 weeks, but we observed fatty liver in CKO mice aged 37 weeks. CKO mice significantly increased liver weight after the 4 weeks of HFD feeding. HFD feeding markedly increased the lipogenesis marker,hepatic TBARS and H2O2 content in CKO mice. On the contrary to this result, the protein expression of the mitochondrial biogenesis factors PGC-1α, pAMPKα and NRF-1 of liver in HFD fed CKO mice. Furthermore HFD feeding led to reduce hepatic mitochondrial aspect ratio, form factor and copy number in CKO mice. Conclusion: These findings suggest that H2O2 production may be an important event triggering NAFLD, and that catalase may be an attractive therapeutic target for preventing NAFLD.