Abstract The health benefits of polyphenols have attracted their use as potential therapeutic agents, food additives, and cosmetics. However, low water solubility of polyphenols limits their cell absorbability, obscuring further exploration. Glycosylation is known to enhance the solubility of polyphenols preserving their pharmacological properties. Here, we show that a uridine diphosphate (UDP) glucosyltransferase from Phytolacca americana ( Pa GT2) regioselectively catalyzes the transfer of glucose from UDP-glucose to stilbenoids such as piceatannol and flavonoids such as kaempferol. To understand the structure-function relationship of Pa GT2, we determined the crystal structure of Pa GT2 as well as Pa GT2 complexed with donor analogue UDP-2-fluoro glucose and stilbenoid acceptor analogues. While only one conserved histidine residue is recognized as a catalytic residue in known UGTs, the crystal structures of Pa GT2 suggested the presence of two catalytically active residues (His18 and His81) at two sides of the catalytic pocket. Although the single catalytic residue mutant His18Ala or His81Ala did not completely impair the glucosylation activity of the enzyme, the double mutant His18Ala/His81Ala failed to form glucoside products. These results showed that both catalytic residues in Pa GT2 actively and independently catalyze glucosylation, hence we called Pa GT2 as an ambidextrous UGT. The information from Pa GT2 will be advantageous for the engineering of efficient biocatalysts for production of therapeutic polyphenols.