Enteropathogenic Escherichia coli (EPEC), a major cause of severe disease with diarrhea in infants, is also involved in weaned rabbit colibacillosis. EPEC 0103 is frequent in rabbit-fattening units of Western Europe. It causes high mortality and growth retardation, leading to substantial economic losses. We report here the construction by allelic exchange of an EPEC 0103 strain mutated in espB and tir, two essential virulence genes. Upon live oral administration to weaned rabbits, the E22DeltaTir/EspB mutant strain efficiently colonized the intestinal tract without any adverse consequences. The rabbits were challenged with the highly pathogenic parental strain E22. The mutant provided complete protection to rabbits and total resistance to intestinal colonization by E22. In addition, E22DeltaTir/EspB strain induced a specific humoral response against the bacterial adhesin AF/R2. These Abs prevent bacterial attachment to epithelial cells in vitro. These results open the way for the development of an efficient vaccine strategy against rabbit EPEC infections.