Enterohemorrhagic E. coli (EHEC) is a human food-borne pathogen responsible for large outbreaks in developed countries. EHEC infections can lead to hemorrhagic colitis and to the life-threatening haemolytic and uremic syndrome mainly through the production of Shiga toxins in the gut.. Contributing to its protective effect against pathogen colonization, the human gut microbiota produces soluble factors that inhibit the expression of Stx2 encoding genes by EHEC. In particular, Bacteroides thetaiotaomicron, a predominant symbiont of the human gut microbiota, was shown to produce such an inhibitory factor. To increase our understanding of the influence of the human gut microbiota on EHEC transcriptome, we investigated the gene expression pattern of EHEC O157:H7 cultivated in the caecal content of germ-free rats and rats associated with the human gut microbiota of a healthy subject or B. thetaiotaomicron alone, using Affymetrix GeneChip E. coli 2.0 genome arrays and qRT-PCR. We showed that the human gut microbiota has a wide effect on EHEC O157:H7 metabolic gene expression by down-regulating several carbohydrate utilization pathways and increasing the expression of genes required for the catabolism of metabolites encountered in the digestive tract. In addition, the human gut microbiota down-regulates a large number of EHEC virulence genes involved in attaching and effacing lesion formation, including genes from the locus of enterocytes effacement (LEE) pathogenicity island and genes encoding non-LEE Type III secreted effectors. Interestingly, we also demonstrated that B. thetaiotaomicron strongly participates to this inhibitory effect. These results show that human gut microbiota and B. thetaiotaomicron negatively influence the expression of many virulence genes in EHEC. This could help developing preventive and therapeutic approaches to limit EHEC gut colonization.