Objectives: In this study, we aimed to study the effect of total flavonoids extracted from Hippophae rhamnoides L. (TFH) on the activity of five CYP450 enzymes in rats by high-performance liquid chromatography (HPLC). We also studied the effect of TFH on the expression of CYP450 enzyme mRNA in rat liver using quantitative real-time PCR (qRT-PCR). Materials and Methods: TFH was orally administered to male rats once daily at doses of 100, 200, and 400 mg/kg/day for 14 days. We established HPLC method to detect the concentration of the analytes in the rat plasma. We used DAS software to calculate the pharmacokinetic parameters. Then, qRT-PCR was used to study the effect of TFH on the expression of CYP450 mRNA. Results: The pharmacokinetic analysis of theophylline in the TFH group revealed a short half-life (t1/2), a decrease in the area under the curve (AUC), and an increase in the value of plasma clearance (CL) (P < 0.05, P < 0.01). In the case of midazolam, t1/2 was prolonged, Cmax was increased (P < 0.05). Results from the PCR analysis indicate that TFH downregulated the expression of CYP3A4 mRNA and upregulated the expression of CYP1A2 mRNA. Conclusion: In conclusion, TFH might induce the activity of CYP1A2 and inhibit the activity of CYP3A4. Herb–drug interaction should be carefully considered when TFH is being used in combination with drugs metabolized by CYP1A2 and CYP3A4.