Methimazole (MMI) is a widely used antithyroid drug, but it can cause hepatotoxicity by unknown mechanisms. Previous studies showed that the hepatic metabolism of MMI produces N-methylthiourea, leading to liver damage. However, the specific enzyme responsible for the production of the toxic metabolite N-methylthiourea is still unclear. In this study, we screened cytochromes P450 (CYPs) in N-methylthiourea production from MMI. CYP2A6 was identified as the key enzyme in catalyzing MMI metabolism to produce N-methylthiourea. When mice were pretreated with a CYP2A6 inhibitor, formation of N-methylthiourea from MMI was remarkably reduced. Consistently, the CYP2A6 inhibitor prevented MMI-induced hepatotoxicity. These results demonstrated that CYP2A6 is essential in MMI bioactivation and hepatotoxicity.