<title>Abstract</title>Brassicaceae plants have a dual-cell type of chemical defense against herbivory. Here, we show a novel single-cell defense involving endoplasmic reticulum (ER)-derived organelles (ER bodies) and the vacuoles. We identify various glucosinolates as endogenous substrates of the ER-body β-glucosidases BGLU23 and BGLU21. Woodlice strongly prefer to eat seedlings of <italic>bglu23 bglu21</italic> or a glucosinolate-deficient mutant over wild-type seedlings, confirming that the β-glucosidases have a role in chemical defense: production of toxic compounds upon organellar damage. Deficiency of the Brassicaceae-specific protein NAI2 prevents ER-body formation, which results in a loss of BGLU23 and a loss of resistance to woodlice. Hence, NAI2 that interacts with BGLU23 is essential for sequestering BGLU23 in ER bodies and preventing its degradation. Artificial expression of NAI2 and BGLU23 in non-Brassicaceae plants results in the formation of ER bodies, indicating that acquisition of NAI2 by Brassicaceae plants is a key step in developing their single-cell defense system.