Enterohemorrhagic (EHEC) is an important foodborne pathogen that infects humans by colonizing the large intestine. The genome of EHEC O157:H7 contains 177 unique O islands (OIs). Certain OIs significantly contribute to the heightened virulence and pathogenicity exhibited by EHEC O157:H7. However, the function of most OI genes remains unknown. We demonstrated here that EHEC O157:H7 adherence to and colonization of the mouse large intestine are both dependent on OI-97. , which is annotated as a LysR-type transcriptional regulator and encoded in OI-97, contributes to this phenotype. Z3495 activated the locus of enterocyte effacement (LEE) gene expression, promoting bacterial adherence. Deletion of significantly decreased the transcription of and other LEE genes, the ability to adhere to the host cells, and colonization in the mouse large intestine. Furthermore, the ChIP-seq results confirmed that Z3495 can directly bind to the promoter region of , which is a well-known activator of Ler, and increase LEE gene expression. Finally, phylogenetic analysis revealed that Z3495 is a widespread transcriptional regulator in enterohemorrhagic and enteropathogenic . As a result of this study, we have gained a deeper understanding of how bacteria control their virulence and provide another example of a laterally acquired regulator that regulates LEE gene expression in bacteria.