Aconitine (AC), an active/toxic alkaloid from Aconitum species, is commonly present in Traditional Chinese Medicine (TCM) prescriptions because of the great effectiveness of Aconitum for the treatment of rheumatoid arthritis, cardiovascular diseases, and tumors in clinic. Buspirone (BP) is a sensitive CYP3A probe drug that is administered through oral/intravenous routes as recommended by the U.S. Food and Drug Administration. This study aims to investigate the influences of AC (0.125 mg/kg, oral) on first-pass (intestinal and hepatic) CYP3A activity by using oral BP as the probe in rats. The pharmacokinetics of oral buspirone hydrochloride at different doses (12.5, 25, and 50 mg/kg) were conducted. The pharmacokinetics of oral BP in rats pretreated with single dose or multiple doses (7-day) of AC were investigated. The plasma concentrations of BP and its major metabolites [1-(2-pyrimidinyl)piperazine (1-PP) and 6'-hydroxybuspirone (6'-OH-BP)] were determined. The formation ratios of 1-PP and 6'-OH-BP from BP (AUC0-∞ of 1-PP/AUC0-∞ of BP and AUC0-∞ of 6'-OH-BP/AUC0-∞ of BP values) showed no alternation when the dose of BP changed. Single dose of AC decreased the AUC0-∞ of BP by 53% but increased the formation ratio of 6'-OH-BP by 74% (P<0.05). Multiple AC exposure increased the AUC0-∞ of BP by 110%, and the formation ratios of 1-PP and 6'-OH-BP from BP were increased by 229% and decreased by 95%, respectively (P<0.05). Conclusively, single/multiple AC exposure did not alter the first-pass CYP3A activity when using oral BP as probe in rats. Nevertheless, multiple AC exposure had markedly changed the production of BP metabolites.