Ampicillin resistance is a marker for hospital-associated Enterococcus faecium. Feces from 208 dogs were selectively screened for the occurrence of ampicillin-resistant E. faecium (AREF). AREF was detected in 42 (23%) of 183 dogs screened in a cross-sectional study in the United Kingdom and in 19 (76%) of 25 dogs studied longitudinally in Denmark. AREF carriage was intermittent in all dogs studied longitudinally. Multilocus sequence typing of 63 canine AREF isolates revealed the presence of 13 distinct sequence types. Approximately 76% of the isolates belonged to hospital-adapted clonal complex 17 (CC17), including those of sequence types ST-78 and ST-192, which are widespread in European and Asian hospitals. Longitudinal screening of 18 healthy humans living in contact with 13 of the dogs under study resulted in the identification of a single, intermittent CC17 carrier. This person carried one of the sequence types (ST-78) recovered from his dog. Based on PCR and Southern hybridization analyses, the putative virulence gene cluster from orf903 to orf907 was widespread in canine AREF isolates (present in 97%), whereas orf2351 (present in 26% of isolates) and orf2430 (present in 31%) were strongly associated with CC17-related sequence types (P < 0.05). Surprisingly, esp and hyl were not detected in any of the isolates. The antimicrobial resistance profiles of canine AREF isolates were not detected in any of the isolates. The antimicrobial resistance profiles of canine AREF isolates generally differed from those previously described for clinical human isolates. The results indicate that dogs are frequent carriers of CC17-related lineages and may play a role in the spread of this nosocomial pathogen. The distinctive virulence and antimicrobial resistance profiles observed among canine AREF isolates raise interesting questions about the origin and evolution of the strains causing human infections.