Aim To investigate the effect of apolipoprotein O knockout mice promoting obesity and metabolic disorders challenged by high fat diet (HFD). Methods Breeding the ApoO heterozygous mice will get the homozygous mice offspring. PCR reaction was used to detect the gene genotype after extracting the mice tissue DNA. mRNA and protein expression of ApoO were determined by quantitative real-time PCR(qRT-PCR) and Western blot assay, respectively. After feeding with high-fat diet, the metabolic phenotypes in mice were observed by somatotype, food intake, lipidomic study in liver. Results The homozygous mice offspring were obtained. It was found that homozygous mice had obesity and more severe hepatic steatosis after HFD challenging. Lipidomics of the liver revealed obvious accumulation of triglyceride and significant changes of phospholipid components. Conclusion The deletion of ApoO may promote obesity and metabolic disorders induced by high-fat diet, which could provide a new target for the prevention and treatment of metabolic syndrome and cardiovascular diseases.