Aim To provide an excellent animal model for the study of atherosclerosis, it generated apolipoprotein E gene knockout (ApoE－/－) rabbit. Methods ApoE gene of New Zealand rabbits were edited and F0 ApoE－/－ rabbits were created by applying CRISPR/Cas9 system. The efficiency and effect of ApoE gene targeted knockout was determined through PCR, TA cloning, Sanger sequencing, Western blot and lipid assay. ApoE＋/－ rabbits were obtained through transmitting by germline. Results Sanger sequencing results showed that ApoE gene was successfully targeted knockout. Serum lipoprotein content of ApoE－/－ rabbits increased in normal diet compared with wild type rabbits and can be transmitted by germline. Conclusion The successful establishment of ApoE－/－ rabbit provided a good animal model for the further study of atherosclerosis and other related diseases.