Objective To study the horizontal transfer mechanism of optrA gene-carrying linezolid-resistant Enterococcus faecalis (LREf) among clinical Enterococcus faecalis mediated by plasmids. Methods Non-repeated LREf from a tertiary first-class hospital in Kunming City from August 2022 to August 2023 were collected and identified by matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing was performed by VITEK 2 Compact, disc diffusion method and microbroth dilution method, optrA gene was detected by polymerase chain reaction (PCR), molecular biology characteristics of LREf was analyzed by whole-genome sequencing (WGS), LREf as the donor strain and clinically isolated Enterococcus faecalis as the recipient strain for conjugation test. Results A total of 17 LREf strains were collected, optrA gene was detected from plasmids of 12 LREf strains. Multiple resistance genes and virulence genes were detected. 12 LREf strains were mainly ST16 type (50.0%). Among the 24 transconjugates in the conjugation test, 8 were successfully conjugated, with a conjugation rate of 33.3%. Further analysis revealed that IS1216E insertion sequences presented at upstream and downstream of the optrA gene on the plasmid before and after conjugation of strains, and formed IS1216E-fexA-optrA-erm (A)-IS1216E-like transposon units. Conclusion The optrA gene can be horizontally transferred among clinical Enterococcus faecalis by mediating of plasmid of genes carrying both erm (A) and fexA, and the insertion sequence IS1216E plays an important role in its horizontal transfer process.