Burkholderia pyrrociniaJK-SH007 can effectively control poplar canker caused by pathogenic fungi. Its antifungal mechanism remains to be explored. Here, we characterized the functional role of CysB inB. pyrrociniaJK-SH007. This protein was shown to be responsible for the synthesis of cysteine and the siderophore ornibactin, as well as the antifungal activity ofB. pyrrociniaJK-SH007. We found that deletion of thecysBgene reduced the antifungal activity and production of the siderophore ornibactin inB. pyrrociniaJK-SH007. However, supplementation with cysteine largely restored these two abilities in the mutant. Further global transcriptome analysis demonstrated that the amino acid metabolic pathway was significantly affected and that some sRNAs were significantly upregulated and targeted the iron–sulfur metabolic pathway by TargetRNA2 prediction. Therefore, we suggest that, inB. pyrrociniaJK-SH007, CysB can regulate the expression of genes related to Fe–S clusters in the iron–sulfur metabolic pathway to affect the antifungal activity ofB. pyrrociniaJK-SH007. These findings provide new insights into the various biological functions regulated by CysB inB. pyrrociniaJK-SH007 and the relationship between iron–sulfur metabolic pathways and fungal inhibitory substances. Additionally, they lay the foundation for further investigation of the main antagonistic substances ofB. pyrrociniaJK-SH007.