AbstractPlant–pest interactions involve multifaceted processes encompassing a complex crosstalk of pathways, molecules, and regulators aimed at overcoming defenses developed by each interacting organism. Among plant defensive compounds against phytophagous arthropods, cyanide-derived products are toxic molecules that directly target pest physiology. Here, we identified the Arabidopsis (Arabidopsis thaliana) gene encoding hydroxynitrile lyase (AtHNL, At5g10300) as one gene induced in response to spider mite (Tetranychus urticae) infestation. AtHNL catalyzes the reversible interconversion between cyanohydrins and derived carbonyl compounds with free cyanide. AtHNL loss- and gain-of-function Arabidopsis plants showed that specific activity of AtHNL using mandelonitrile as substrate was higher in the overexpressing lines than in wild-type (WT) and mutant lines. Concomitantly, mandelonitrile accumulated at higher levels in mutant lines than in WT plants and was significantly reduced in the AtHNL overexpressing lines. After mite infestation, mandelonitrile content increased in WT and overexpressing plants but not in mutant lines, while hydrogen cyanide (HCN) accumulated in the three infested Arabidopsis genotypes. Feeding bioassays demonstrated that the AtHNL gene participated in Arabidopsis defense against T. urticae. The reduced leaf damage detected in the AtHNL overexpressing lines reflected the mite’s reduced ability to feed on leaves, which consequently restricted mite fecundity. In turn, mites upregulated TuCAS1 encoding β-cyanoalanine synthase to avoid the respiratory damage produced by HCN. This detoxification effect was functionally demonstrated by reduced mite fecundity observed when dsRNA-TuCAS-treated mites fed on WT plants and hnl1 mutant lines. These findings add more players in the Arabidopsis–T. urticae interplay to overcome mutual defenses.