Objective To prepare a Bama minipig model of hereditary tyrosinemia type III, thehydroxyphenylpyruvate dioxygenase (Hpd) gene was chosen to be edited by the CRISPR/ Cas9 technology. Methods Templates (used as in vitro transcripts for Cas9 mRNA and sgRNA) were amplified from pST1374-NLS-flag-linker-Cas9and pGL3-U6-gRNA-PGK-puromycin by PCR, respectively, and subsequently transcribed in vitro into Cas9 mRNA andsgRNA-Hpd. Finally, cas9 mRNA and sgRNA-Hpd were co-injected into the cytoplasm of single cell embryos to generatean Hpd-modified Bama minipig. Results Twenty embryos of Bama minipigs co-injected with cas9 mRNA and sgRNA-Hpdwere transplanted into two pseudopregnant mothers. We obtained four offsprings with a modified Hpd gene. Conclusions Inthis study, we have successfully generated Hpd knockout Bama minipigs with hereditary tyrosinemia type III using the CRISPR/ Cas9 technique, which will be a valuable model for research of the tyrosine metabolic pathway.