Objective：To investigate the relationship between pilA and hylEfm genes and the pathogenic mechanism of enterococcus fae－cium. Methods：A multiplex PCR was applied to detect the distribution of hylEfm and pilA. The transfer mode between strains of ente－rococcus faecium was confirmed via filter matings. S1 nuclease and I-CeuI assay was performed to detect the position of pilA and hylEfm genes. Results：Totally 137 strains of enterococcus faecium were isolated，88% clinical isolates（120/137 isolates）carrying pilA genes and 15% clinical isolates（21/137 isolates）carrying hylEfm gene. Fifty strains of inpatients and 50 strains of health controls were isolated respectively；70% isolates（35/50 isolates） carrying pilA genes and 52% isolates（26/50 isolates） carriying pilA genes were respectively observed in inpatients and health controls. Highly significant differences in isolates carrying pilA genes among clinical group，inpatients group and health controls group were observed（P<0.000 1，P=0.008 0）. Filter mating confirmed that pilA and hylEfm genes can transfer between different strains through plasmid conjugation. Southern hybridization found both pilA and hylEfm genes located at a large plasmid with size running between 145.5 kb and 291 kb. Conclusion：Positive rate of Pilus-like structures protein genes pilA of enterococcus faecium isolated from our hospital is consistently high. pilA and hylEfm genes simultaneously encode on a large plasmid containing variable antibiotic resistance genes in different isolates of nosocomial enterococcus faecium strains.