Escherichia coli is a microorganism that plays an important role in digestion and nutrient absorption and belongs to intestinal flora. However, under certain conditions, it can become pathogenic and cause various infections, including urinary tract infections, gastroenteritis and sepsis. It has been demonstrated that certain E. coli strains can lose characteristic biochemical properties, including motility and sugar fermentation. Identification of these strains poses a considerable challenge for microbiology laboratories. Our case presents a lactose-negative, non-motile E. coli strain causing a urinary tract infection in a 62-year-old male patient with bladder carcinoma. For bacterial identification, traditional biochemical tests (sugar fermentation, motility, ornithine, lysine, indole tests), a fully automated bacterial identification test (Phoenix, BD, USA), a semi-automated bacterial identification test (API 20E, bioMérieux, France), specific antisera, and MALDI-TOF MS (MALDI Biotyper Sirius, Bruker Daltonics, Bremen, Germany) identification system were used. The analyses revealed a lactose-negative, non-motile E. coli strain that exhibited phenotypic characteristics. In the present study, we examined a lactosenegative, non-motile E. coli strain that caused a urinary tract infection in order to highlight these challenging phenotypes.