AbstractThe domestication of plants has commonly resulted in the loss of plant defense metabolites, with important consequences for the plants' interactions with herbivores and their natural enemies. Squash domestication started 10′000 years ago and has led to the loss of cucurbitacins, which are highly toxictriterpenes. The banded cucumber beetle (Diabrotica balteata), a generalist herbivore, is adapted to feed on plants from the Cucurbitaceae and is known to sequester cucurbitacins, supposedly for its own defense. However, the evidence for this is inconclusive. In this study we tested the impact of squash domestication on the chemical protection ofD. balteatalarvae against a predatory rove beetle (Dalotia coriaria). We found that cucurbitacins do not defend the larvae against this common soil dwelling predator. In fact,D. balteatalarvae were less attacked when they fed on cucurbitacin‐free roots of domesticated varieties compared to high‐cucurbitacin roots of wild plants. This study appears to be the first to look at the consequences of plant domestication on belowground tritrophic interactions. Our results challenge the generalized assumption that sequestered cucurbitacins protect this herbivore against natural enemies, and instead reveals an opposite effect that may be due to a tradeoff between coping with cucurbitacins and avoiding predation.