Cardenolides are potent plant defensive metabolites that have been studied for decades for their significance in plant-insect interactions and their use in treating heart failure in humans. With recent advancements in genome and transcriptome sequencing, genes in the cardenolide biosynthetic pathway have begun to be identified. Here we employed gene co-expression network analysis using published data from the cardenolide-producing plant Erysimum cheiranthoides (wormseed wallflower) to identify two UDP-dependent glycosyltransferases, UGT73C44 and UGT73C45, that are capable of glucosylating the aglycone cardenolide digitoxigenin, as well as other predicted cardenolide pathway intermediates. In vitro and in planta assays revealed that UGT73C44 acted on cardenolide pathway intermediates with a low Km value of 7.0 μM for digitoxigenin, while UGT73C45 displayed broader substrate specificity in vitro and could glucosylate diverse steroid and flavonoid substrates. A phylogeny and comparisons of structural models of UGT73C44 and UGT73C45 suggest that the enzymes have divergent active site architectures, which may account for their different substrate specificities. These data report the first plant-derived UGT specific to cardenolides, advancing our understanding of cardenolide biosynthesis and the enzymes that drive specialized metabolite diversity. These findings lay the foundation for future efforts to reconstitute the cardenolide pathway in heterologous systems and design cardenolide analogs with the potential for improved therapeutic properties.