This study aimed to evaluate the acaricidal activity of lettuce (Lactuca sativa) extracts against carmine spider mites (Tetranychus cinnabarinus Boisd.) and isolate the acaricidal components. Acaricidal activities of lettuce extracts isolated from different parts (the leaf, root and seed) using various solvents (petroleum ether, acetone and methanol) were evaluated with slide-dip bioassay and relatively high median lethal concentration (LC50) values were detected. Acetone extracts of lettuce leaves harvested in July and September were fractionated and isolated with silica gel and thin-layer chromatography. Consequently, acetone extracts of lettuce leaves harvested in July exhibited higher acaricidal activity than those harvested in September, with an LC50 value of 0.268 mg ml−1 at 72 h post-treatment. A total of 27 fractions were obtained from the acetone extract of lettuce leaves harvested in July, and mite mortalities with the 11th and 12th fractions were higher than those with the other 25 fractions (LC50: 0.751 and 1.258 mg ml−1 at 48 h post-treatment, respectively). Subsequently, active acaricidal components of the 11th fraction were identified by infrared, nuclear magnetic resonance and liquid chromatography/mass spectrometry. Five components were isolated from the 11th fraction, with components 11-a and 11-b showing relatively high acaricidal activities (LC50: 0.288 and 0.114 mg ml−1 at 48 h post-treatment, respectively). Component 11-a was identified as β-sitosterol. In conclusion, acetone extracts of lettuce leaves harvested in July might be used as a novel phytogenic acaricide to control mites.