C-glycosyltransferases (CGTs) are important enzymes that are responsible for the synthesis of the C-glycosides of flavonoids and isoflavonoids. Flavonoid CGTs have been molecularly characterized from several plant species; however, to date, no gene encoding an isoflavonoid CGT has been reported from any plant species. A significant example of an isoflavonoid C-glycoside is puerarin, a compound that contributes to the major medicinal effects of Pueraria lobata. Knowledge regarding the C-glucosylation that occurs during puerarin biosynthesis remains scant. One possible route for the puerarin synthesis is via the C-glucosylation of daidzein. This study describes the molecular cloning and functional characterization of a novel glucosyltransferase (PlUGT43) from P. lobata. Biochemical analyses revealed that PlUGT43 possesses an activity for the C-glucosylation of daidzein to puerarin; it shows activity with the isoflavones, daidzein and genistein, but displays no activity towards other potential acceptors, including flavonoids. To validate the in vivo function of PlUGT43, the PlUGT43 gene was over-expressed in soybean hairy roots that naturally synthesize daidzein but that do not produce puerarin. The expression of PlUGT43 led to the production of puerarin in the transgenic soybean hairy roots, confirming a role of PlUGT43 in puerarin biosynthesis.