Objective The aim of this study was to establish F0 generation chimeric rabbits with FOXN1 gene knockout to explore methods for in vivo conservation of immunodeficient rabbits in a conventional housing environment. Methods Initially, the CRISPR/Cas9 technology was employed to inject the constructed sgRNA and Cas9 protein into a single cell of rabbit two-cell stage embryos to obtain chimeric embryos with FOXN1 gene editing. Subsequently, the embryos were transferred into surrogate does. Finally, the F0 generation offspring 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 results confirmed the successful establishment of chimeric rabbits with FOXN1 gene knockout. Observation revealed that the chimeras exhibited normal growth and development in a conventional environment without any immunodeficient phenotypes. Conclusion This study has initially established a chimeric rabbit model with FOXN1 gene knockout, which can grow and develop normally in ordinary environments. This lays the foundation for further breeding of FOXN1 immunodeficient rabbits in the future.