Many animals protect themselves from predation with chemicals, both self-made or sequestered from their diet. The potential drivers of the diversity of these chemicals have been long studied, but our knowledge them, and their acquisition mode, is heavily based on specialist herbivores that sequester their defences. The wood tiger moth (Arctia plantaginis, Linnaeus, 1758) is a well-studied aposematic species, but the nature of its chemical defences has not been fully described. Here we report the presence of two methoxypyrazines, 2-sec-butyl-3-methoxypyrazine and 2-isobutyl-3-methoxypyrazine, in the moths’ defensive secretions. By raising larvae on an artificial diet, we confirm, for the first time, that their defensive compounds are produced de novo rather than sequestered from their diet. Pyrazines are known for their defensive function in invertebrates due to their distinctive odour, inducing aversion and facilitating predator learning. While their synthesis has been suspected, it has never previously been experimentally confirmed. Our results highlight the importance of considering de novo synthesis, in addition to sequestration, when studying the defensive capabilities of insects and other invertebrates.