Objective To evaluate in vitro antimicrobial activity of tedizolid(TZD) against Enterococcus faecalis(E. faecalis), and explore antimicrobial resistance mechanism and distribution of multilocus sequence typing (MLST) of tedizolid-non-susceptible E. faecalis. Methods E. faecalis isolates from Nanshan People's Hospital of Shenzhen City between January 1, 2011 and June 30, 2016 were collected, antimicrobial resistance of isolates was tested by automatic instrument method and micro-broth dilution method. Polymerase chain reaction (PCR) was used to detect the carrying status of oxazolidinone antibiotic resistance genes, isolated strains were typed by MLST. Results A total of 289 strains of E. faecalis were obtained, strains were mainly from department of surgery (57.4%) and main specimen source was midstream urine (126 strains, 43.6%). Susceptibility rate of 289 E. faecalis strains to TZD was 94.1%, susceptibility rates to ampicillin, furacillin and vancomycin were all high (97.9%-99.7%). MLST results showed that there were 47 ST types, the dominant ST were ST16 and ST179, accoun-ting for 29.1% (84 strains) and 24.9% (72 strains) respectively, among TZD-non-susceptible E. faecalis, the proportion of ST16 was higher than that of ST179 (P<0.05). A total of 17 strains of TZD-non-susceptible E. faecalis strains were isolated, the proportion of optrA gene carried by them was higher than that of susceptible strains. Conclusion TZD is superior to linezolid in antimicrobial activity against E. faecalis, but it has no good antimicrobial activity against E. faecalis carrying optrA gene.