Abstract  [Aim]  To investigate the development of resistance to diafenthiuron and the mechanism responsible for this resistance, in Tetranychus urticae. [Methods]  A susceptible population of T. urticae was continuously selected for resistance to diafenthiuron for 33 generations in a laboratory. The activity of carboxylesterases (CarEs), glutathione S-transferases (GSTs), Mixed-function oxidases (MFOs) and chitinases (chitinases) in this population was tested every six generations. [Results]  The resistance index had reached 18.61 after 33 generations. The activity of CarEs, GSTs and MFOs in the resistant population were significantly higher (1.909, 2.119 and 7.436-fold, respectively) than in the sensitive population (P < 0.05). However, there was no significant difference in the chitinase activity in the sensitive populations (0.594, P < 0.05). [Conclusion]  Resistance of T. urticae to diafenthiuron is mainly associated with MFOs and chitinase activity, although GSTs and CarEs also play a role.