Bacterial pathogens exploit intricate sensory mechanisms to precisely modulate gene expression in response to host-associated cues. This optimises within-host fitness. D-serine, a host-produced metabolite enriched in urine is toxic to enterohaemorrhagic E. coli, inhibiting its type 3 secretion system (T3SS) and activating the SOS response. While this has been proposed to restrict EHEC to its preferred intestinal niche, the molecular mechanism of virulence repression by D-serine remains incomplete. Here we show that multiple amino acids, including L-serine, converge on this same regulatory pathway and repress the T3SS but without triggering the SOS response. A combination of transcriptomics, metabolomics, and targeted deletions reveal that this regulation is mediated by the release of ammonia, the nitrogenous product of serine breakdown, rather than by sensing of intact serine. Interestingly, despite evolutionary loss of canonical D-serine metabolic capacity, EHEC shows a novel oxidative deamination activity capable of producing this regulatory signal. Deamination has the effect of switching off the nitrogen stress response in T3SS-inducing media as seen by repression of the entire regulon of NtrC – the nitrogen stress response master regulator. Deletion mutants lacking ntrC were unable to fully activate the T3SS and showed no repression of T3SS genes by L- or D-serine. Our data suggest that distal intestinal colonisation by EHEC is facilitated by adaptation of virulence factor regulation to amino acid-depleted environments. This work highlights the crucial interplay between stress responses, metabolism and virulence in an important bacterial pathogen.