Enterohaemorrhagic Escherichia coli (EHEC) are bacterial pathogens responsible for life-threatening diseases in humans such as bloody diarrhoea and the hemolytic and uremic syndrome. To date, no specific therapy is available and treatments remain essentially symptomatic. In recent years, we demonstrated in vitro that nitric oxide (NO), a major mediator of the intestinal immune response, strongly represses the synthesis of the two cardinal virulence factors in EHEC, namely Shiga toxins (Stx) and the type III secretion system, suggesting NO has a great potential to protect against EHEC infection. In this study, we investigated the interplay between NO and EHEC in vivo using mouse models of infection. Using a NO-sensing reporter strain, we determined that EHEC sense NO in the gut of infected mice. Treatment of infected mice with a specific NOS inhibitor increased EHEC adhesion to the colonic mucosa but unexpectedly decreased Stx activity in the gastrointestinal tract, protecting mice from renal failure. Taken together, our data indicate that NO can have both beneficial and detrimental consequences on the outcome of an EHEC infection, and underline the importance of in vivo studies to increase our knowledge in host-pathogen interactions.