Abstract: Pueraria thomsonii has long been used in traditional Chinese medicine. Isoflavonoids are the principle pharmacologically active components, which are primarily observed as glycosyl-conjugates and accumulate in P. thomsonii roots. However, the molecular mechanisms underlying the glycosylation processes in (iso)flavonoid biosynthesis have not been thoroughly elucidated. In the current study, an O-glucosyltransferase (PtUGT8) was identified in the medicinal plant P. thomsonii from RNA-seq database. Biochemical assays of the recombinant PtUGT8 showed that it was able to glycosylate chalcone (isoliquiritigenin) at the 4-OH position and glycosylate isoflavones (daidzein, formononetin, and genistein) at the 7-OH or 4′-OH position, exhibiting no enzyme activity to flavonones (liquiritigenin and narigenin) in vitro. The identification of PtUGT8 may provide a useful enzyme catalyst for efficient biotransformation of isoflavones and other natural products for food or pharmacological applications.