Cyanogenic plants can release hydrogen cyanide (HCN) by degrading the cyanogenic glycosides with β-glucosidase, which plays an important role in plant defense. Studies have demonstrated the HCN-mediated toxicity against pests by a plant-delivered strategy, however, the individual toxicity of HCN was not well-demonstrated due to the interference of other metabolites. Besides, some sap-sucking pests can detoxify HCN, but to what extent can the pests sustain the toxicity of HCN remained enigmatic. To address these questions, the two-spotted spider mite (TSSM), and its cyanogenic hosts, which comprised a TSSM-resistant (C1115) and a TSSM-susceptible (KU50) cassava cultivars, were used as models. Feeding preference assay illustrated that TSSM was inclined to settle on the middle-bottom leaves of KU50, since the linamarin and the HCN doses were significantly lower compared with C1115 and top leaves. Fumigation using leaf extract indicated that mortality derived from C1115 was higher than KU50, and inhibiting β-glucosidase caused the decline of HCN release and non-lethal effect on TSSM. Moreover, reproduction and development were suppressed when TSSMs were exposed to sub-lethal dose of HCN. Our findings speculated that HCN shape cassava resistance against TSSM in a threshold-dependent manner, which offers insights for developing novel resistant variety with high cyanogenic capacity.