Objectives To characterize a glycosyltransferase (UGT74AN3) from Catharanthus roseus and investigate its specificity toward cardiotonic steroids and phenolic compounds. Results UGT74AN3, a novel permissive GT from C. roseus, displayed average high conversion rate (> 90%) toward eight structurally different cardiotonic steroids. Among them, resibufogenin, digitoxigenin, and uzarigenin gave 100% yield. Based on LC-MS, H-1-NMR and C-13-NMR analysis, structure elucidation of eight glycosides was consistent with 3-O-beta-D-glucosides. We further confirmed UGT74AN3 was permissive enough to glycosylate curcumin, resveratrol, and phloretin. The cDNA sequence of UGT74AN3 contained an ORF of 1,425 nucleotides encoding 474 amino acids. UGT74AN3 performed the maximum catalytic activity at 40 degrees C, pH 8.0, and was divalent cation-independent. K-m values of UGT74AN3 toward resibufogenin, digitoxigenin, and uzarigenin were 7.0 mu M, 12.3 mu M, and 17.4 mu M, respectively. Conclusions UGT74AN3, a glycosyltransferase from a noncardenolide-producing plant, displayed catalytic efficiency toward cardiotonic steroids and phenolic compounds, which would make it feasible for glycosylation of bioactive molecules.