Xenocoumacin 1 (Xcn1), a major antimicrobial compound produced by Xenorhabdus nematophila, has great potential for use in agricultural productions. In this study, we evaluated the effects of CpxR, a global response regulator associated with the mutualism and pathogenesis of X. nematophila, on the antimicrobial activity and Xcn1 production. The mutation of cpxR could promote the production of Xcn1 significantly with its level in Delta cpxR mutant being 3.07 times higher than that in the wild type. Additionally, the expression levels of xcnA-L genes, which are responsible for the production of Xcn1, were increased in Delta cpxR mutant while the expression levels of xcnMN, which are required for the conversion of Xcn1 into Xcn2 was reduced. Noticeably, Xcn2 was also enhanced on account of the conversion of excessive Xcn1 in spite of low expression levels of xcnM and xcnN in Delta cpxR mutant. The transcriptional levels of ompR and lrp, encoding the global response regulators OmpR and Lrp which negatively and positively regulate the production of Xcn1 were concurrently decreased and increased, respectively. Correspondingly, Delta cpxR mutant also exhibited increased antimicrobial activities in vitro and in vivo. Together, these findings suggest that CpxR negatively regulates xcnA-L genes expression while positively regulating xcnMN expression in X. nematophila YL001, which led to a high yield of Xcn1 in Delta cpxR mutant.