The attachment of enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) to host intestinal epithelial cells is essential for the development of hemorrhagic colitis and hemolytic-uremic syndrome in humans. Genes involved in attachment are carried within a pathogenicity island named the locus of enterocyte effacement (LEE), known to be directly activated by quorum sensing (QS). In the present study, we investigated autoinducer-2 (AI-2) production and the expression of several virulence-related genes in EHEC O157 grown in the absence and presence of a Lactobacillus acidophilus-secreted molecule(s). Transcription of important EHEC O157 virulence-related genes was studied by constructing promoter-reporter fusions and reverse transcriptase PCR. Shiga toxin (Stx) production was assayed by an enzyme immunoassay. When EHEC O157 was grown in the presence of chromatographically selected fractions of L. acidophilus La-5 cell-free spent medium, we observed a significant reduction of both extracellular AI-2 concentration and the expression of important virulence-related genes, although no significant difference in Stx production was observed. We show here that L. acidophilus La-5 secretes a molecule(s) that either acts as a QS signal inhibitor or directly interacts with bacterial transcriptional regulators, controlling the transcription of EHEC O157 genes involved in colonization.