Objective: Apolipoprotein E (Apoe) plays a crucial role in lipid metabolism. The CRISPR/Cas9 was utilized to generate an Apoe knockout mouse model for further investigation the role of Apoe in lipid metabolism and atherosclerosis. Method: Two sgRNA designed for the Apoe in C57BL/6J mice were co-injected with Cas9 mRNA into fertilized eggs, followed by transplantation into ICR recipient mice to obtain F0 generation mice. Positive mice were identified by PCR screening of extracted tail DNA. The expression of Apoe mRNA in various tissues of mice was assessed by quantitative real-time PCR, while lipid indexes were measured in serum samples of mice. The lipid accumulation in the inner lining of aortic vessel was detected by Oil red O staining. Result: PCR and sequencing results confirmed the successful construction of Apoe knockout mice (C57BL/6-Apoeem1/Nifdc, referred to as Apoe KO); the results of quantitative real-time PCR exhibited that the expression of Apoe mRNA was significantly reduced in the liver, brain, spleen, kidney, and lung tissues of the Apoe KO homozygote mice (Apoe-/-). Additionally, the elevated serum total cholesterol and low-density lipoprotein cholesterol levels were occurred in Apoe-/- mice, and lower high-density lipoprotein cholesterol levels were also occurred in males. Compared to wild-type mice, the extensive lipid plaques in the inner lining of arteries were appeared in the Apoe-/- mice under normal chow consumption conditions. Conclusion: This study was successfully established an Apoe knockout mouse model exhibiting typical abnormal lipid metabolism phenotype with arterial lipid accumulation even without a high-fat diet intervention. This work provides background data for the Apoe KO mouse resource and a new model for the study of abnormal lipid metabolism phenotype.