Objective To establish F0 generation chimeric rabbits with FOXN1 gene knockout and explore method for the in vivo conservation of immunodeficient rabbits in a conventional housing environment. Methods Initially,CRISPR/ Cas9 technology was employed to inject constructed sgRNA and Cas9 protein into a single cell from rabbit two-cell stage embryos to obtain chimeric embryos with FOXN1 gene editing. The embryos were subsequently transferred into surrogate does. Finally, the F0 generation offsprings were genotyped using PCR and Sanger sequencing, and their growth and development in a conventional housing environment were observed. Results The PCR and Sanger sequencing result confirmed the successful establishment of chimeric rabbits with FOXN1 gene knockout. On observation, the chimeras exhibited normal growth and development in a conventional environment without any immunodeficient phenotypes. Conclusions This study established a preliminary chimeric rabbit model with FOXN1 gene knockout that grows and develops normally in standard laboratory environments. This lays the foundation for the further breeding of FOXN1 immunodeficient rabbits in the future.