Like other members of the Brassicaceae, plants in the wallflower genus (Erysimum) produce glucosinolates, which are potent defenses against a wide range of herbivores. As a more recently evolved second line of defense, Erysimum produces cardiac glycosides, which are allosteric inhibitors of Na+,K+-ATPases in animals. Cardiac glycoside biosynthesis has evolved in diverse lineages including foxglove (Digitalis, Plantaginaceae) and milkweeds (Apocynaceae), but the full biosynthetic pathway has not been described in any species. We identify and generate CRISPR/Cas9 knockouts of two cytochrome P450 monooxygenases involved in cardiac glycoside biosynthesis in wormseed wallflower (Erysimum cheiranthoides L.): EcCYP87A126, which cleaves the side chain from sterol precursors to initiate cardiac glycoside biosynthesis, and EcCYP716A418, which has a role in cardiac glycoside hydroxylation. In the EcCYP87A126 knockout lines, cardiac glycoside production is eliminated, effectively reversing Erysimum’s escape from herbivory. For the generalist herbivores green peach aphid (Myzus persicae Suzler) and cabbage looper (Trichoplusia ni Hübner), cardiac glycosides appear to be largely redundant with glucosinolates, having some effect in choice assays but little to no effect on insect performance. By contrast, the crucifer-feeding specialist cabbage butterfly (Pieris rapae L.), which will not oviposit or feed on wildtype E. cheiranthoides, is able to complete its life cycle on cardenolide-free E. cheiranthoides mutant lines. Thus, our study demonstrates in vivo that cardiac glycoside production allows Erysimum to escape from a specialist herbivore.