Ethanolamine (EA) metabolism is a trait associated with enteric pathogens, including enterohemorrhagic Escherichia coli O157:H7 (EHEC). EHEC causes severe bloody diarrhea and hemolytic uremic syndrome. EHEC encodes the ethanolamine utilization (eut) operon that allows EHEC to metabolize EA and gain a competitive advantage when colonizing the gastrointestinal tract. The eut operon encodes the transcriptional regulator EutR. Genetic studies indicated that EutR expression is induced by EA and vitamin B12 and that EutR promotes expression of the eut operon; however, biochemical evidence for these interactions has been lacking. We performed EA-binding assays and electrophoretic mobility shift assays (EMSAs) to elucidate a mechanism for EutR gene regulation. These studies confirmed EutR interaction with EA, as well as direct binding to the eutS promoter. EutR also contributes to expression of the locus of enterocyte effacement (LEE) in an EA-dependent manner. We performed EMSAs to examine EutR activation of the LEE. The results demonstrated that EutR directly binds the regulatory region of the ler promoter. These results present the first mechanistic description of EutR gene regulation and reveal a novel role for EutR in EHEC pathogenesis.