Intestinal colonization by antibiotic-resistant Enterococcus faecium is the first step in a process that can lead to infections in hospitalized patients. By comparative genome analysis and subsequent polymerase chain reaction screening, we identified a locus that encodes a putative phosphotransferase system (PTS). The PTS locus was widespread in isolates from hospital outbreaks of infection (84.2%) and nonoutbreak clinical infections (66.0%) but absent from human commensal isolates. Deletion of pstD, which is predicted to encode the enzyme IID subunit of this PTS, significantly impaired the ability of E. faecium to colonize the murine intestinal tract during antibiotic treatment. This is the first description of a determinant that contributes to intestinal colonization in clinical E. faecium strains.