The effects of Bifidobacterium longum BB536 (BB536) on Escherichia coli O157:H7 IPH9 (IPH9) were first examined using germ-free mice mono-associated with BB536 (BB536-MA). Thirty-two days after oral challenge with a lethal dose of IPH9, all the non-associated germ-free mice (GF) died, but all the BB536-MA mice survived and the number of IPH9 in the feces was lower than that in GF mice. Second, co-culture experiments with BB536 and IPH9 were performed. The number of IPH9 in the co-culture was less than 1% of that in mono-culture after 24 hr, and the verotoxin concentration also decreased. Culture of IPH9 under various conditions showed that lactate, acetate and the supernatant of BB536 culture had anti-O157 activities even when the pH was neutral, and the supernatant of BB536 culture had stronger inhibitory effects than lactate or acetate against the production of verotoxin. This efficient inhibition of verotoxin may suggest the presence of verotoxin-inhibitory factors in BB536 metabolites in addition to lactate and acetate.