Despite the monarch butterfly (Danaus plexippus) being famous for its adaptations to the defensive traits of its milkweed host plants, little is known about the macroevolution of these traits. Unlike most other animal species, monarchs are largely insensitive to cardenolides, since their target site, the sodium pump (Na+/K+-ATPase), has evolved amino acid substitutions which reduce cardenolide binding (so-called target site insensitivity, TSI). Since many, but not all, species of milkweed butterflies (Danaini) are associated with cardenolide-containing host plants, we analyzed 17 species, representing all phylogenetic lineages of milkweed butterflies, for the occurrence of TSI by sequence analyses of the Na+/K+-ATPase gene and by enzymatic assays with extracted Na+/K+-ATPase. Here we report that sensitivity to cardenolides was reduced in a stepwise manner during the macroevolution of milkweed butterflies. Strikingly, not all Danaini typically consuming cardenolides showed TSI, but rather TSI was more strongly associated with sequestration of toxic cardenolides. Thus, the interplay between bottom-up selection by plant compounds and top-down selection by natural enemies can explain the evolutionary sequence of adaptations to these toxins.