Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen that specifically colonizes and infects the human large intestine. EHEC O157:H7 engages intricate regulatory pathways to detect host intestinal signals and regulate virulence-related gene expression during colonization and infection. However, the overall EHEC O157:H7 virulence regulatory network in the human large intestine remains incompletely understood. Here, we report a complete signal regulatory pathway where the EvgSA two-component system responds to high-nicotinamide levels produced by microbiota in the large intestine and directly activates loci of enterocyte effacement genes to promote EHEC O157:H7 adherence and colonization. This EvgSA-mediated nicotinamide signaling regulatory pathway is conserved and widespread among several other EHEC serotypes. Moreover, disruption of this virulence-regulating pathway by the deletion of evgS or evgA significantly decreased EHEC O157:H7 adherence and colonization in the mouse intestinal tract, indicating that these genes could be potential targets for the development of new therapeutics for EHEC O157:H7 infection.