Aim: To evaluate whether berberine hydrochloride (BBR) could modify the pharmacokinetic profiles of midazolam (MDZ), a substrate of CYP3A, and rhodamine 123 (Rh123), a substrate of P-glycolprotein (P-gp), in male rats. Methods: The rats were given with varied does of BBR or 75 mg/kg ketoconazole as a positive control for 10 days by intragastric administration. Single-pass duodenum perfusion of 20 mg/kg MDZ and inguinal artery canulated rats were used in the study. Plasma concentrations of MDZ and 1′-hydroxymidazolam (1′-OH-MDZ) were analyzed by high performance liquid chromatography (HPLC). The rats were given with varied does of BBR or 4 mg/kg verapamil as a positive control for 10 days by intragastric administration. Blood was obtained from the caudal vein of rats after single-pass intragastric administration of 5 mg/kg Rh123. HPLC was used to analyze the plasma concentrations of Rh123. Results: BBR produced similar results as the ketoconazole (positive control group) with a dose-dependent increase in the AUC and AUMC of midazolam except at the dose of 50 mg/kg ( p < 0.01). And BBR could significantly increase the peak plasma concentrations (C) of MDZ ( p < 0.01), but reduce the clearance rate (CL) and the apparent volume of the distribution (V) of MDZ ( p < 0.05). The results also indicated that BBR had no significant impact on the half-life period (t) and the time to reach peak concentration (t). Meanwhile, BBR could dose-dependently decrease AUC and AUMC of 1′-OH-MDZ significantly ( p < 0.05), and expedite the clearance rate of 1′-OH-MDZ while gaining its apparent volume of distribution ( p < 0.05), but had no significant impact on t and T. The result also showed that BBR, except at the dose of 50 mg/kg, and the positive verapamil group could significantly increase the AUC and AUC of Rh123 ( p < 0.001), meanwhile raise C of Rh123 and shorten its V inversely ( p < 0.05). Additionally, pre-treatment with BBR had no significant influence with the half-life period of Rh123, while significantly reduced its clearance rate ( p < 0.05). Conclusion: The metabolism of MDZ and Rh123 was controlled by BBR. The results were most likely due to the inhibition by BBR on CYP3A enzymes and P-gp transporter. [ABSTRACT FROM AUTHOR]