Cyanogenic glucosides (CNglcs) are phytoanticipins that are present in more than 2650 plant species. They play an important role in defense against generalist herbivory due to their bitter taste and their hydrolysis upon tissue disruption results in release of toxic hydrogen cyanide. Zygaena larvae (Zygaenidae, Lepidoptera) both sequester (take up and accumulate) the CNglcs linamarin and lotaustralin from their food plants (Fabacea) and biosynthesize them de novo from valine and isoleucine. The presented research demonstrates that de novo biosynthesis of CNglcs in Z. filipendulae is dependent on the CNglc levels in the food plant. Tight regulation of the de novo biosynthesis, both on the transcript and the enzyme steady-state level, allows precise control of ratios and content of CNglcs. De novo biosynthesis is shown to take place in close proximity to the storage chambers in the skin which enables rapid and continuous refill after usage in predator defense. During pupation, de novo biosynthesis is shut down and CNglcs slowly turned over. De novo biosynthesis of CNglcs in adult females is demonstrated and localized to the skin, while adult males unexpectedly do not de novo biosynthesize CNglcs. Adult females are shown to transfer CNglcs to the eggs following emergence from the pupa, providing protection against predation at this vulnerable developmental stage. It is finally speculated that de novo biosynthesis of CNglcs enabled colonization of and subsequently sequestration from the food plant, and has been maintained for fine-tuning of CNglc levels and composition for an optimized defense capacity.