【Objective】 Cloning the potential nucleoside antibiotic gene cluster from Xenorhabdus szentirmaii DSM 16338 and establishment the genetic manipulation system for X. szentirmaii DSM 16338. 【Method】 Cloning of the target gene cluster was done by direct PCR amplification of different segments that were joined together, and the in vitro homologous recombination of different segments was fulfilled by using a native yeast recombination system. Meanwhile, the sacB gene of fructan-sucrose transferase was employed as a negative selection marker to construct gene knockout mutant strains through double homologous arm recombination for establishing genetic manipulation system forX. szentirmaii DSM 16338. 【Result】 The predicted target gene cluster was successfully obtained. Large fragment deletion mutant strain designated as GW2 was constructed by knocking out three consecutive genes of this predicted gene cluster. In order to avoid the regulatory gene inhibiting the transcription expression of the entire predicted gene cluster in the negative regulatory process, the regulatory gene knockout mutant strain GW4 was selected. 【Conclusion】 A target gene cluster in X. szentirmaii DSM 16338 was obtained by using an efficient direct cloning method. The gene cluster was also interrupted and the genetic manipulation system of Xenorhabdus szentirmaii DSM 16338 was constructed successfully. This study will provide a foundation for further studies on the biological and chemical diversity of natural products produced by bacteria of the genus, Xenorhabdus.