Objective The high-frequence mutant genotype Condons41 / 42 (-CTTT) of human thalassaemia- related HBB gene ( hHBB) was knocked into genome of the porcine fibroblasts by using CRISPR-Cas9 Site-specific recombination systems. Then the transfected fibroblasts were screened to establish homozygous cell lines with positive genotype. Methods (1) Synthesize the human HBB mutant with Condons41 / 42 (-CTTT) and cloned the left and right homology arm located at the flanking sequence of the pig HBB gene. (2) Then the HBB mutant was inserted between the left and right homology arm using infusion PCR method to building a final homologous recombination vector. ( 3) The sgRNA targeting pig HBB gene were designed using online software and then were verified its activity in pig PK15 cells. (4) Bama pig fetal fibroblasts were co-transfected with CRISPR/ Cas9 vector, sgRNA and pGH-LA-hHBB-RA using by electroporation and then were screened to establish single clone. Results (1) We synthesized full length of the hHBB CDS with a mutant Condons41 / 42 (-CTTT). The left and right homology arm of 1. 062 kb and 1. 024 kb were cloned from Bama pig genome. Then we constructed a final vector pGH-LA-hHBB-RA. ( 2) Two sgRNAs targeted pig HBB gene were validated in PK15 cells and used for further experiments. (3) Finally, we identified 15 single-cell clones of the porcine fibroblast carry the human HBB gene mutant. Conclusions We generated porcine fibroblast carrying the human HBB gene mutant using CRISPR/ Cas9. Our study provides key materials for scientist to generate pig models of human thalassemia.