The autoinducer-2 (AI-2) molecule is produced by many bacterial species, including various human gastrointestinal (GI) tract commensal bacteria, and has been proposed to be involved in interspecies communication. Because pathogens are likely to encounter AI-2 in the GI tract, we studied the effects of AI-2 on various phenotypes associated with enterohemorrhagic Escherichia coli (EHEC) infections. AI-2 attracted EHEC in agarose plug chemotaxis assays and also increased swimming motility, as well as increased EHEC attachment to HeLa cells. The molecular basis underlying the stimulation of EHEC chemotaxis, motility, and colonization by AI-2 was investigated at the transcriptome level using DNA microarrays. We found that exposure to AI-2 altered the expression of 23 locus of enterocyte effacement (LEE) genes directly involved in the production of virulence determinants, as well as other genes associated with virulence (e.g., 46 flagellar/fimbrial genes, 24 iron-related genes), in a temporally defined manner. To our knowledge, this is the first study to report AI-2-mediated regulation of EHEC chemotaxis and colonization, as well as temporal regulation of EHEC transcriptome by AI-2. Our results suggest that AI-2 is an important signal in EHEC infections of the human GI tract.