OBJECTIVES: The poxtA gene is a transferable linezolid-resistance gene that encodes an ATP-binding cassette F protein that prevents linezolid from inhibiting protein synthesis. In enterococci, the presence of the poxtA gene does not consistently imply resistance to linezolid. The objective of this work was to analyze the role of the poxtA gene in linezolid susceptibility in a cohort of five poxtA METHODS: Three of the isolates were linezolid-resistant and two were linezolid-susceptible. The genomes of all five isolates were sequenced using short and long read sequencing. The genomes were assembled to identify the location of the poxtA gene. The presence and relative amount of the PoxtA protein was determined with a proteomics approach. RESULTS: One of the linezolid-resistant isolates harbored a deletion in the poxtA gene promoter and a mutation in the ribosomal protein L4. Another exhibited two sets of tandem repeats of the poxtA gene within the chromosome, and the third displayed an increased copy number of the plasmid carrying the poxtA gene. Proteomic analysis detected the PoxtA protein and confirmed increased expression levels in the three resistant mutants. The highest expression was seen in the promoter deletion mutant. CONCLUSION: The presence of the poxtA gene in clinical isolates of E. faecium does not imply resistance to linezolid, but it should be considered a significant risk factor for the development of resistance. Active molecular surveillance for intestinal poxtA gene carriers could be important to prevent dissemination.