The bovine gastrointestinal tract (GIT) is the main reservoir for enterohaemorrhagic Escherichia coli (EHEC) responsible for food-borne infections. Therefore, it is crucial to develop strategies, such as EHEC suppression by antagonistic microorganisms, to reduce EHEC survival in the GIT of cattle and to limit shedding and food contamination. Most human-derived Lactobacillus reuteri strains produce hydroxypropionaldehyde (HPA), an antimicrobial compound, during anaerobic reduction of glycerol. The capacity of L. reuteri LB1-7 to produce HPA and its antimicrobial activity against EHEC FCH6 were evaluated in bovine rumen fluid (RF) under strict anaerobiosis. EHEC was totally suppressed when incubated in RF inoculated with L. reuteri LB1-7 and supplemented with 80 mM glycerol. The addition of LB1-7 or glycerol alone did not modify EHEC survival in RF. Glycerol was converted to HPA (up to 14 mM) and lactate (30 mM) by LB1-7, but only HPA production seemed to be responsible for EHEC suppression. Furthermore, L. reuteri LB1-7 did not have major adverse effects on ruminal digestion of common feedstuffs. The bactericidal activity of L. reuteri LB1-7, the concentration of glycerol required and the level of HPA secreted depended on physiological and ecological environments. In vitro experiments also showed that EHEC inoculated in rumen fluid and exposed to L. reuteri and glycerol had a very limited growth in rectal contents.