1. We investigated whether bergamottin would be useful for evaluating CYP3A-mediated intestinal metabolism in rats utilising its characteristics as a mechanism-based inhibitor of CYP3A.2. Buspirone and fexofenadine, probe substrates of CYP3A and P-glycoprotein (P-gp), respectively, were orally co-administered to rats with bergamottin (2.5 mg/kg) or orally administered 2 h after bergamottin pre-treatment. The effect of bergamottin pre-treatment on hepatic CYP3A specifically was investigated with intravenous administration of buspirone. The kobs of bergamottin for CYP3A was calculated based on the portal unbound Cmax.3. Co-administration of bergamottin significantly increased the AUC0–inf for buspirone and fexofenadine by 1.6-fold and 1.7-fold, respectively, indicating that bergamottin inhibited both CYP3A and P-gp.4. Bergamottin pre-treatment significantly elevated the AUC0–inf of oral buspirone by 3.7-fold but exerted no effect on the pharmacokinetics of intravenous buspirone, indicating that bergamottin pre-treatment selectively inhibited CYP3A-mediated intestinal metabolism without affecting the hepatic CYP3A. These findings were supported by the result that the kobs (0.00000118 min−1) of bergamottin for CYP3A was lower than the kdeg (0.0005 min−1) for CYP3A. Furthermore, bergamottin pre-treatment did not affect the pharmacokinetics of oral fexofenadine, suggesting that P-gp was not influenced.5. These profiles of bergamottin enable the convenient assessment of CYP3A-mediated intestinal metabolism.