Tiki1 gene, found and named by Professor He Xi"s team from Boston Children"s Hospital, Harvard Medical School, plays a key role in the formation of head in Xenopus. However, as Tiki1 gene is absent in rodents such as mice, it is impossible to use mice or rat to study its role in mammals. In this study, we generated Tiki1 gene modified pigs using CRISPR/Cas9 system combined with somatic cell cloning technology to study the role of Tiki1 gene in pig development. Aligned the human Tiki1 mRNA sequence provided by professor He Xi"s team with the pig genome database, we selected two target sites (g1 and g2) with the top 2 highest sequence identity at the predicted pig Tiki1 gene locus. The sgrRNA plasmid was constructed to transfect porcine fetal fibroblasts, and 52 single cell clones were screened and sequenced. We finally selected 5 single-cell clones with biallelic knockout mutations at target site g1 and 3 single-cell clones with biallelic knockout mutations at target site g2 as nuclear donors for constructing Tiki1 knockout pigs. A total of 720 recombinant embryos were constructed and transferred into three surrogate sows and one of them was successfully pregnant monitored by B ultrasound. A total of 13 cloned piglets (ten living piglets and 3 dead piglets ) were produced, and 12 of them were biallelic knockout mutations at Tiki1 locus. Both the living and dead Tiki1 gene knockout cloned piglets were developed normally and the living piglets have survived healthy till now. The results indicate that the role of Tiki1 gene on early development of pigs is different from that of frogs. The specific role of Tiki1 gene in the early development of pigs needs to be further investigated.