Acequinocyl is the only commercialized acaricide of the naphthoquinone analogue group. To develop strategies to minimize resistance, a laboratory-selected acequinocyl-resistant strain of the two-spotted spider mite Tetranychus urticae was used to determine the inheritance, toxicological, biochemical and cross-resistance data. The LC50 and LC60 values of acequinocyl were determined on T. urticae using a leaf-disc method and spraying tower. A laboratory selection population designated ACE6 had a122.0-fold resistance to acequinocyl following six selection cycles. A selected strain showing 112.0-fold resistance was named ACE6 population. This population developed low resistance to etoxazole and spirodiclofen and moderate cross-resistance to hexythiazox, milbemectin and bifenazate. Monooxygenase enzyme inhibitor piperonyl butoxide, the esterase enzyme inhibitor S-benzyl O, O-diisopropyl phosphorothioate and the glutathione S-transferase (GST) enzyme inhibitor diethyl maleate synergized resistance values were 1.0-, 2.5- and. < 1-fold, respectively. Crossing experiments revealed that acequinocyl resistance in the ACE6 population was intermediately dominant and monogenic. In addition, the detoxifying enzyme activities were increased 3.03-fold for esterase,. < 1-fold for GST and 1.00-fold for cytochrome P450 monooxygenase in the ACE6 population. The results suggest that esterase enzyme may play a role in acequinocyl resistance, while the GST and P450 enzymes do not appear to have any significant involvement.