An increased incidence of enrofloxacin-resistant Escherichia coli associated with septicemic colibacillosis in calves was observed recently in northern Italy. The aim of this study was to investigate this phenomenon. A total of 47 consecutive E. coli isolates exhibiting reduced susceptibility to enrofloxacin (intermediately resistant or resistant) causing septicemic colibacillosis in calves from 45 large-scale farms during 2006-2008, were studied. Phylogenetic group, antimicrobial agents susceptibility, and O serogroup were determined with randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) typing, providing additional discrimination. All of the microorganisms carried resistance to two or more additional drugs, with the pattern fluoroquinolone-ampicillin-co-trimoxazole-tetracycline-gentamicin-thiamphenicol being the most represented (18/47; 38.3%). Plasmid-mediated extended-spectrum and AmpC β-lactamases and plasmid-mediated fluoroquinolone resistance genetic determinants were not detected. Third-generation cephalosporins emerged as the most active antimicrobial agents tested (97.9% of susceptible strains). Overall, 37 different RAPD profiles and 18 different O serogroups could be distinguished among the typeable strains, indicating a substantial heterogeneity and suggesting the occurrence of several independent selection events. However, approximately one-fourth (11/47) of the strains belonged to serogroup O78, and PFGE revealed that the great majority (7/11) of these were clonally related, indicating the selection of a O78 clonal group. This is the first report investigating the molecular epidemiology of fluoroquinolone-resistant E. coli in calves and describing the emergence of a fluoroquinolone-resistant E. coli clonal group in these animals.