Aims: This study was prompted to investigate the intestinal localization and colonization of orally administered Escherichia coli Nissle 1917 (EcN) in piglets. Methods and Results: EcN was fed to ten EcN-negative piglets (3 months) over seven consecutive days. Faecal samples were collected repeatedly and tested for EcN-DNA by a combined culture/PCR assay and for viable EcN by culture methods, respectively. EcN-DNA was detectable in faeces of all piglets within the first 24 h after it was added to the feed. After the administration of EcN had been stopped, the presence of EcN-DNA in faecal samples indicated that all piglets shedded EcN with their faeces intermittently through up to 33 days. In addition, E. coli strains indistinguishable from EcN by all markers tested (rdar colony morphotype, multiplex PCR and GEI II-PCR analyses, XbaI-pattern, K5 phage susceptibility) were isolated from faecal samples and from mucosal swabs taken at euthanasia at the end of the experiment. Conclusions: EcN colonizes the intestine and persists in conventionally reared piglets for at least 4 weeks upon oral administration. Significance and Impact of the Study: Results of this study have implications for efficacy and safety assessments of EcN as a probiotic strain for use in pigs.