Abstract: Recombinant protein of vancomycin glycosyltransferase, GtfE, was obtained by gene cloning and hete-rologous expression. Gene gtfE was amplified from the genomic DNA of vancomycin producing strain and ligated into expression vector pET-37b, the recombinant plasmid GtfE/pET-37b was transformed into E. coli BL21(DE3). The product of in vitro glucosylation reaction catalyzed by purified GtfE was isolated and identified by HPLC and ESI-MS, respectively. The assay indicated that the recombinant protein GtfE had expected glucosylation activity to transfer uridine 5′-diphosphoglucose to the vancomycin aglycone. This study laid the foundation for producing glycosyl diversity of vancomycin.