The objective of this study was to evaluate the ability of schisandrin A (SchA) to inhibit the P450 enzyme CYP3A in vivo. Male Sprague-Dawley rats were intragastrically administered with varied doses of SchA (8 mg/kg or 16 mg/kg or 32 mg/kg) or 75 mg/kg ketoconazole for three consecutive days. Ketoconazole, a chemical inhibitor of CYP3A, was used as positive control. Subsequently, changes in hepatic microsome CYP3A activity and the pharmacokinetic profiles of midazolam (MDZ), a specific CYP3A substrate, were studied as indicators of rat hepatic microsomal activity of CYP3A. Differences in the plasma concentrations of MDZ and its related metabolites and the hepatic microsome concentrations of 1'-hydroxymidazolam were analysed by high-performance liquid chromatography. The current results provide direct and explicit evidence that SchA produced concentration-dependent inhibition of MDZ metabolite formation in rat liver microsomes (p < 0.01 or p < 0.001). Regular SchA consumption also caused concentration-dependent increase in Cmax and area under the concentration-time curve (AUC0-t and AUC0-∞ ) of peroral MDZ (p < 0.05 or p < 0.01) compared to vehicle-treated rats, whereas those of its metabolites (1'-hydroxymidazolam) were reduced (p < 0.05 or p < 0.01). Analysis of the data suggests that changes in the pharmacokinetic profiles of peroral MDZ in the rat model were contributed mainly to SchA inhibition of CYP3A activity. These results suggest that SchA, as an inhibitor of CYP3A, possesses a clinically beneficial property of altering the disposition of drugs metabolized by CYP3A.