Glycosyltransferases (GTs) are key enzymes for the post-modification of secondary metabolites in drug development processes. In our prior research, an one-pot enzymatic system produced alpha-mangostin 3,6-di-O-beta-D-glucopyranoside (Mg1) at a higher proportion using wild-type glycosyltransferase (YjiC) but alpha-mangostin 3-O-beta-D-glucopyranoside (Mg2) exhibited markedly higher anti-bacterial activities. This study focuses on a Bacillus licheniformis-originated flexible glycosyltransferase by mutagenesis to examine the active site residues involved in glycosylation for a product specificity towards Mg2. The generated H298A, H298S, and H298C mutants of YjiC exhibited a regiospecificity towards glycosylated product (Mg2) and were targeted in this study. The production pattern of Mg1 decreased to 63 (H298A), 85 (H298S) and 95% (H298C) yields compared to the wild-type YjiC. The increase of uridine 5'-diphosphate (UDP) leading to the inhibition of enzyme activity and production of uridine 5'-diphosphate glucose (UDP-glucose) in overall system was critical for the specific glycosylated product formation rate. H298A, H298S, and H298C mutants and YjiC exhibited 244, 251, and 186% increases in Mg2 production yields, respectively. And also H298A, H298S, and H298C showed 281, 279, and 251% increases in yield of Mg3 compared with wild type YjiC, respectively. There was improved conversion of both mono-glucosides product (Mg2a and Mg3) than di-glucosides products. The H298 mutants were found to overcome the limitation of the wild-type YjiC for regioselective synthesis of Mg2 by an enzymatic system.