Host-derived metabolites regulate bacterial physiology and contribute to infection dynamics. D-serine, abundant in extraintestinal environments but scarce in the gut, serves as a niche-specific signal influencing E. coli pathotype adaptation and virulence. In enterohaemorrhagic E. coli (EHEC), a major foodborne pathogen, D-serine exposure impairs growth, but the mechanisms underlying this toxicity remain unclear.  Transcriptome datasets were analysed to identify differentially expressed genes in response to D-serine. Growth inhibition was evaluated across minimal media with diverse carbon sources and rescue assays with specific metabolite supplementation were performed. Deletion of sgrS was used to assess its role in D-serine toxicity. Further testing with the SgrS mutant was used to monitor activity of the known D-serine targets, LEE1 and RecA.  The small RNA SgrS, canonically linked to glucose-phosphate stress, was identified as the most strongly upregulated transcript. However, growth inhibition remained unaffected across diverse carbon sources, including those independent of SgrS-regulated transport. Deletion of sgrS did not restore growth nor did supplementation with pyruvate or pantothenate, while L-serine provided partial rescue, implicating disruption of fundamental metabolic processes. Reporter assays confirmed D-serine-induced repression of LEE1 and induced SOS response activity occurs independently of SgrS. D-serine elicits a pathotype-specific response in EHEC that disrupts central metabolism and stress regulation. While not directly responsible for growth inhibition, the strong induction of SgrS reflects indirect activation of stress pathways and reveals a broader transcriptomic reprogramming than previously recognised. These findings demonstrate how host-derived metabolites can trigger diverse regulatory networks and expose unique vulnerabilities in pathogenic E. coli.