10-Hydroxyl mesaconitine (10-OH MA) is a new potential toxicity marker of Radix Aconiti Lateralis Preparata (Fuzi), which is widely used for the treatment of cardiovascular diseases, rheumatoid arthritis, and other illnesses. Recently, we found that 10-OH MA concentrations in the plasma and urine of outpatients are comparable to those of aconitine, one of the most important toxicity markers of Fuzi. Published studies have shown that CYP3A plays important roles in the disposition and toxicity of Fuzi. This study aims to elucidate the role of cytochrome P450 enzymes in the metabolism and toxicity of 10-OH MA. 10-OH MA was transformed to 14 and 12 metabolites when incubated with human and mouse liver microsomes, respectively. The major metabolic pathways included hydroxylation, dehydrogenation, and demethylation. Human recombinant enzyme metabolic assays revealed that CYP3A5 was mainly involved in 10-OH MA metabolism, followed by CYP3A4, CYP3A7, CYP1A2, and CYP2C8. CYP3A inhibitor ketoconazole significantly inhibited the metabolism of 10-OH MA in vitro. The dose-normalized area under the plasma concentration-time curve from time 0 to t (AUC/Dose) and tissue 10-OH MA concentrations were significantly increased in Cyp3a-inhibited C57BL/6J mice administered with 10-OH MA, which was accompanied by a sharp decrease in the LD value and enhanced cardiotoxicity and neurotoxicity. Moreover, diltiazem markedly increased the AUC/Dose of 10-OH MA by 17.6-fold. Additionally, 10-OH MA competitively inhibited CYP3A5 and CYP3A4 in vitro, with apparent K values of 5.0 and 13.6 μmol/L, respectively. In conclusion, CYP3A5 and CYP3A4 are the pivotal enzymes responsible for the metabolism and toxicity of 10-OH MA. Coexposure to 10-OH-MA and CYP3A inhibitors likely significantly increased 10-OH MA toxicity. SIGNIFICANCE STATEMENT: 10-Hydroxyl mesaconitine (10-OH MA) is a potential toxicity marker of Radix Aconiti Lateralis Preparata (Fuzi), the metabolism of which is predominantly mediated by CYP3A5 and CYP3A4. CYP3A inhibition markedly increases systemic exposure and cardiotoxicity/neurotoxicity of 10-OH MA. In addition, 10-OH MA competitively inhibits CYP3A, indicating the risks of coadministration with CYP3A inhibitors.