Aim: To investigate the inhibitory interactions of bufalin and CYP3A4.Methods: Recombinant human CYP3A4 was incubated with bufalin in vitro. Bufalin was administered ig and iv to Wistar rats to further estimate its impact on CYP3A4, and midazolam was given to index the activity of CYP3A4. Results: The IC50 of bufalin was 14.52 μmol/L. Bufalin affected CYP3A4 activity with increases in AUC0-t and t1/2f and decreases in CL and the formation of 1-hydroxy-midazolam after ig or iv administration of midazolam (P<0.05). An increase in Cmax after ig bufalin administration (P<0.05) was observed.Conclusion: Bufalin showed a modest but significant inhibition of CYP3A4 both in vitro and in vivo. The likelihood of an interaction between bufalin and the CYP3A4-metabolized drugs in human might not be negated.