Abstract: Pseudomonas protegens H78 can produce multiple broad-spectrum antibiotics such as pyoluteorin（Plt）. The biosynthesis of these antibiotics is controlled by the complex regulatory mechanism and network. This study aims to ascertain the regulatory mechanism of Plt biosynthesis by two homologous small RNA（sRNA） genes crcY and crcZ in the carbon catabolism repression regulatory system（Cbr/Crc） of strain H78. Firstly, the crcY/Z dual mutant was constructed by the non-scar deletion technology. The fermentation analysis result showed that Plt biosynthesis was almost completely inhibited by the crcY/Z dual deletion in H78. Secondly, the gene complementary experiment showed the complementation of crcY or crcZ gene could restore Plt biosynthesis of the crcY/Z dual mutant. Finally, the lacZ reporter analysis result further confirmed that the crcY/Z deletion significantly down regulated the expression of Plt biosynthetic operon pltLABCDEFG. These results demonstrated that the sRNA genes crcY/Z had a significant positive regulatory effect on the Plt biosynthesis and its gene expression in P. protegens. This study will lay the foundation for further elucidating the regulatory network of Plt biosynthesis and improving Plt production through metabolic engineering.