The effect of itraconazole, a potent inhibitor of the CYP3A isoenzyme family, on the pharmacokinetics of imidafenacin, a novel synthetic muscarinic receptor antagonist, was investigated. Twelve healthy subjects participated in this open-label, self-controlled study. In period I, subjects received a single oral dose of 0.1 mg imidafenacin. In period II, they received multiple oral doses of 200 mg itraconazole for 9 days and a single oral dose of 0.1 mg imidafenacin on day 8. Plasma concentrations of imidafenacin and M-2, the major metabolite of imidafenacin metabolized by CYP3A4, were determined. Analytes were measured by liquid chromatog-raphy tandem mass spectrometry. Following coadministra-tion with itraconazole, the maximum plasma concentration (Cmax) of imidafenacin increased 1.32-fold (90% confidence intervals [CIs]: 1.12–1.56), and the area under the plasma concentration-time curve from time 0 to infinity (AUC0-∞) increased 1.78-fold (90% CI: 1.47–2.16). In conclusion, itraconazole increases the plasma concentrations of imidafenacin by inhibiting CYP3A4. Therefore, itraconazole or potent CYP3A4 inhibitors should be carefully added to imidafenacin drug regimens.