Abstract: To conduct the characterization of its pharmacokinetics in rats of nifedipine sustained-release pellets and to study the relationship between the pellets and CYP3A4 activity. A gradient HPLC method was developed to simultaneously determine 6β-hydroxycortisol and hydrocortisone. CYP3A4 activity of rats was quantified by urinary ratio of 6β-hydroxycortisol/hydrocortisone after intravenous injection of hydrocortisone as a biomarker. HPLC method was also developed to quantify the drug concentration in plasma of rats, and the studies of pharmacokinetics were performed after oral administration of single dose of two formulations: Nifedipine matrix sustained-release pellets and nifedipine tablet(using as control). The results showed that the ratio of ten rats was 0. 271±0. 129. c_max of nifedipine sustained-release pellets decreases by nearly 70%, t_max significantly increased by 400% and t_1/2 and MRT significantly increased by 230% compared to control. Nifedipine sustained-release pellets had a significant sustained-release property compared to the control and CYP3A4 activity affected its pharmacokinetics behavior.