The presence of hyl gene in 364 PFGE clones of Enterococcus faecium was detected by colony hybridization under conditions of high stringency. The isogenic hyl-deficient mutant (* hyl) was constructed with suicide pTX4577 and screened by allelic replacement. Moreover, an in vitro study was made on the effect of hyl gene detection on the growth ability of hylgene detection on the mutant, and an in vivo study was made on the decrease of virulence in the mouse peritonitis model. The results showed, in the clinical isolates, the positive percentage of hyl gene was 32.8%, which was significantly higher than that (5.3%) in the non-clinical isolates. The * hyl was selected by kanamycin and identified by PCR, pulsed field gel electrophoresis (PFGE) and Southern blot. The experimental evidence indicated that the growth ability of * hyl was remarkably reduced in comparison with that of the wild-type strain. The percentage survival of mice in TX2466 groups was 0, while that of * hyl groups was 50% at the same inoculum in mouse peritonitis. The differences were significant. These data suggest that hyl gene in specific E. faecium strains may be enriched in determinants that make them more likely to cause clinical infections. Being important in the pathogenesis, hyl gene is probably a major virulence factor of Enterococcus faecium.