Escherichia coli Nissle 1917 (EcN) is a well-characterized probiotic bacterium. Although genomic comparisons of EcN with the uropathogenic E. coli strain CFT073 revealed high degrees of similarity, EcN is generally considered a non-pathogenic organism. However, as recent evidence suggests that EcN is capable of inducing inflammatory responses in host intestinal epithelial cells, we aimed to investigate potential pathogenic properties of EcN in an in vivo model using various germ-free (GF) mouse strains. With the exception of C3H/HeJZtm mice, which carry a defective toll-like receptor (TLR)4-allele, no lesions were obvious in mice of different strains orally inoculated with EcN for 1 week, although organ cultures (blood, lung, mesenteric lymph node, pancreas, spleen, liver and kidney) tested positive to various degrees. C3H/HeJZtm mice inoculated with EcN became clinically ill and the majority died or had to be euthanized. Organs of all gnotobiotic C3H/HeJZtm mice were positive for EcN by culture; major histological findings were moderate to severe pyogranulomatous serositis, typhlitis and pancreatitis. Histological findings were corroborated by highly elevated tumour necrosis factor (TNF) serum levels. Lesions were not detected in specified pathogen free maintained C3H/HeJZtm mice, GF C3H/HeJ mice lacking the interleukin-10 gene, or GF C3H/HeJZtm mice that were inoculated with E. coli K12 strain MG1655 as a control. In addition, mild histological lesions were detected in Ztm:NMRI mice 3 months after oral inoculation with EcN. This study shows that EcN is capable of displaying a virulent phenotype in GF C3H/HeJZtm mice. Whether this phenotype is linked to the bacterium's probiotic nature should be the focus of further studies.