Objective To establish the heterologous recombinant embryonic stem cell （ES cell） with ghrelin receptor (GHS-R) gene deletion in order to study the function of the GHS-R gene. Methods PGK-neo cassette was replaced by TK-neo in X-pPNT agent. The target region was located in exon 1 and exon 2 of GHS-R. Two homologous arms were amplified from mouse ES cells genomic DNA and constructed into X-pPNT with SalⅠ/NotⅠand EcoRⅠ/BamHⅠ, respectively. ES cells were electrotransfected with the linearized targeting vector and screened with G418 and Gancyclovir. Finally, the positive ES cell clones were identified by PCR and sequencing. Results The X-pPNT-TK-neo vector was obtained. And two homologous arms were inserted correctly. Finally, 328 positive clones were obtained by G418 and Gancyclovir screening, and 3 clones were confirmed as GHS-R gene homologous recombination. Conclusion This study provides the necessary basis for the establishment of the GHS-R knock out mouse model and the further study on GHS-R gene function in vivo.