The defences of aposematic animals are characterised by diversity and variability of secondary metabolites. Here we examine the nature and function of chemical defence diversity in large milkweed bugs, Oncopeltus fasciatus, testing the hypothesis that different chemical defence compounds have evolved in response to different enemies. We profiled and quantified the cardenolides sequestered by large milkweed bugs in their defensive secretions and their bodies, and measured the inhibitory properties of a subset of isolated milkweed cardenolides in the insect’s defence against the Na+/K+—ATPase target site of vertebrate and invertebrate predators, using porcine Na+/K+—ATPase data as a reference. We show that highly concentrated coroglaucigenin cardenolides in the insect’s defence (glucopyranosyl frugoside and frugoside) are toxic for both resistant and sensitive predators, whereas corotoxigenin and calotropagenin cardenolides have varying degrees of enzyme inhibition among various predators. Overall, O. fasciatus is well defended against a range of enemies due to the differential effect of these compounds´ target sites. Our results suggest that the compounds the insect sequester have evolved in response to predation pressure.