Obesity and obesity-associated fatty liver disease (FLD) are becoming global health problems (Evans et al. 2004). Hepatic triglyceride (TG) accumulation is considered to be a prerequisite for developing FLD. Peroxisomes are single membrane-bound organelles, which have many important functions in lipid metabolism, including fatty acids β-oxidization. Pex11α is one of Pex11 gene factor was shown to promote peroxisome division (Delille et al. 2010, Koch et al. 2010). The pathogenic link between obesity and FLD and peroxisome biogenesis remains unclear. To understand the molecular and physiological functions of the Pex11α gene, we disrupted this gene in mice. Five-week old Pex11α-/- (KO) mice and matched C57BL/6 (WT) mice were fed a high-fat diet (HFD) for 12 weeks. Body weights in KO mice were significantly higher than those in WT mice fed a normal diet or an HFD. The amount of food intake was similar between KO mice and WT mice. Oxygen consumption was significantly lower in KO mice than WT mice. Weights of Epididymal white adipose tissue (e-WAT) of KO mice were significantly greater than WT mice. Large adipocytes were more prevalent in KO mice. Fatty acid synthase and peroxisome proliferator-activated receptor γ mRNA levels were significantly higher in the brown adipose tissues of KO mice than in WT mice by approximately ten- and two-fold. Hepatic TG concentrations in KO mice were significantly higher than those in WT mice fed a normal diet or an HFD. We also characterized the response to 24-hr fasting. Hepatic TG concentrations in fasted Pex11α-/- mice were significantly higher than in fasted WT mice. Very low density lipoprotein production test showed that plasma TG levels increased at higher rates in WT mice than in KO mice after treatment with 500mg/kg of lipoprotein lipase inhibitor tyloxapol. By both catalase (a peroxisome matrix protein) and PMP70 (a peroxisome membrane protein) immunofluorescence staining, the number of peroxisomes was lower in the livers of KO mice than in those of WT mice. Ultrastructural analysis showed that small and regular spherical peroxisomes were more prevalent in KO mice fed normal chow supplemented without or with 0.015% fenofibrate (w/w) for 2 days. We observed a significantly higher ratio of empty peroxisomes containing only PMP70 but not catalase in KO mice. Immunoblotting results also showed a significantly lower ratio of catalase and PMP70 proteins in the peroxisomes of KO mice after fenofibrate treatment. mRNA expression levels of peroxisomal fatty acid oxidation-related genes (ATP-binding cassette, sub-family D, member 2 and acyl-CoA thioesterase 3) were significantly higher in WT mice than in KO mice. Our results provide the first evidence that Pex11α deficiency impairs peroxisome elongation and abundance and peroxisomal fatty acid oxidation, thus contributing to increased lipid accumulation in the liver and increased de novo lipogenesis in adipose tissue.