Calotropin is commonly stored as a defense mechanism by insects that eat milkweeds as their main food source.

In the pregnane pathway, a plant analog of the mitochondrial cytochrome P450 (CYP11A in humans), is thought to catalyze the conversion of pregnenolone to progesterone.

Calotropagenin may be produced by the same process as digitoxigenin, however the mechanism for production of calotropin and digitoxin from these genins diverges.

[3][5] Inhibition of the Na+/K+-ATPase causes an increase of sodium inside the cell, and by the action of the sodium-calcium exchanger (NCX) also raises the calcium concentration.

Calotropin has a more noticeable effect on the myocardium than it does on skeletal muscles, as these cells have more active NCX proteins.

[7] Poisoning can cause a variety of unpleasant symptoms including abdominal pain, bloating, diarrhoea, muscle tremors, seizures, and death.

[12] The insects primarily retain calotropin and calactin (a configurational isomer), even though other cardenolides exist in the plant latex and leaves.

Monarch butterflies also store several volatile pyrazines, which give off an odor, which is a warning signal to predators to avoid the insect.