Abstract: The aim of this study was to determine and analyze the DNA sequence of the endogenous plasmid pXW from Enterococcus faecalis EXW27, and to construct an Escherichia coli/Enterococcus faecalis shuttle vector based on its minimal replicon. The endogenous plasmid pXW was isolated from E. faecalis EXW27 with good probiotic properties. The DNA sequence of pXW was determined and analyzed, and then the replicon of plasmid pXW was used to construct an Escherichia coli/Enterococcus faecalis shuttle vector. The host range, transformation efficiency and stability of the Escherichia coli/Enterococcus faecalis shuttle vector were also studied. The results showed that plasmid pXW was 8617 bp in size and its GC content was 33.29%. It contained 8 ORFs and was assumed to be θ-type replicating plasmid. The copy number of plasmid pXW in E. faecalis EXW27 was up to 32.09±0.93, indicating that pXW was a high copy number plasmid. In this study, the minimal replicon of plasmid pXW was determined, and an Escherichia coli/Enterococcus faecalis shuttle vector was constructed based on this replicon. The shuttle vector had a wide host range and was successfully transformed into different types of lactic acid bacteria. The transformation efficiency was between 1.96×10²~8.96×10⁴ CFU/μg (plasmid DNA), and the plasmid loss rate was between 28.54% and 54.17%. In this study, we successfully constructed an Escherichia coli/Enterococcus faecalis shuttle vector with wide host range, high transformation efficiency and high stability, which provides a new tool for gene manipulation of lactic acid bacteria.