Background <p>We previously identified a regulator NsdA_sr, which negatively regulated rimocidin biosynthesis in <i>Streptomyces rimosus</i> M527. However, the exact regulatory mechanism of NsdA_sr on rimocidin production remains unknown.</p> Results <p>In this study, firstly, transcriptomic data demonstrated that the differentially expressed genes resulting from the over-expression of <i>nsdA</i>_<i>sr</i> were primarily associated with several key metabolic pathways, including glycolysis, oxidative phosphorylation, and ribosome-related genes, all of which were downregulated. This directly impacted the concentrations of CoA and NADH, as confirmed by concentration measurement assays. Subsequently, the results of the ChIP-seq experiments revealed that NsdA_sr directly binds to 49 target genes. Notably, these include <i>RS18275</i> and <i>RS18290</i> (both involved in fatty acid degradation) as well as <i>rpoB</i> (related to DNA transcription). The validity of the ChIP-seq assay for these three genes was further supported by in vitro electrophoretic mobility shift assays. Regarding <i>RS18275</i> and <i>RS18290</i>, the results revealed that the binding of NsdA_sr to these elements led to the downregulation of gene expression. This, in turn, resulted in a decrease in the levels of butyryl-CoA and malonyl-CoA, which are known precursors for rimocidin biosynthesis. Consequently, this negatively impacted on the biosynthesis of rimocidin. In the case of <i>rpoB</i>, the results indicated that NsdA_sr binding led to a downregulation of overall protein levels. This was determined by enzymatic activity of report gene GUS and Western blot assay. Consequently, this resulted in a decrease in rimocidin yield.</p> Conclusion <p>This study reveals NsdA_sr’s dual role in limiting rimocidin production by suppressing metabolic precursors and modulating protein expression. Integrated transcriptomic and ChIP-seq analyses provide critical insights into its regulatory mechanisms.</p>