Type IV bundle-forming pili of enteropathogenic Escherichia coli are required for the localized adherence and autoaggregation phenotypes. Whether these pili are also required for virulence was tested in volunteers by inactivating bfpA or bfpT(perA) encoding, respectively, the pilus subunit and thebfp operon transcriptional activator. Both mutants caused significantly less diarrhea. Mutation of the bfpFnucleotide-binding domain caused increased piliation, enhanced localized adherence, and abolished the twitching motility–dispersal phase of the autoaggregation phenotype. The bfpF mutant colonized the human intestine but was about 200-fold less virulent. Thus, BfpF is required for dispersal from the bacterial aggregate and for full virulence.