ABSTRACT Background Thegene polymorphisms of the CYP2C9, as well as the substrate specificity of theenzyme, result in different clearances for different substrates by CYP2C9variants. Research designand methods The CYP2C9 wild type and 38 CYP2C9 variants, expressed in insectmicrosomes, were incubated with azilsartan. The resulting metabolite,O-desethyl azilsartan, was determined by HPLC-MS/MS. The enzyme kineticparameters of the 38 variants were calculated and compared with the wild type.Subsequently, we selected CYP2C9 × 1, *2, and * 3 as target proteins for moleculardocking with azilsartan to elucidate the mechanisms underlying changes inenzyme function. Results Comparedwith CYP2C9 × 1, three variants (CYP2C9 × 29, *39, and * 49) exhibited markedlyincreased CLint values (from 170%-275%, *p < 0.05), whereas 28variants exhibited significantly decreased CLint values (from3%-63%,*p < 0.05). The molecular docking results showed that the binding energy ofCYP2C9 × 2 and * 3 was lower than that of the wild type. Conclusion Thisassessment revealed the effect of CYP2C9 gene polymorphisms on azilsartanmetabolism, establishing a theoretical basis for further in-vivo studies andclinical applications. This study will help expand the database of CYP2C9gene-drug pairs and identify appropriate treatment strategies for azilsartan,contributing to the field of precision medicine.