The object of this paper was to study the in vitro percutaneous factors that affect permeability of imperatorin. Excised abdominal skin of SD rats was used as the permeation model. Modified Franz diffusion cell was used to evaluate receiving solution, skin treatment and drug concentration on the effect of permeation. HPLC was established to determine drug content in recetpor at specified time points. The result showed that 20% ethanol and 0.5% Tween-80 could significantly increase the steady-state infiltration rate (Jass) of imperatorin. Skin treated of 20% ethanol, 20% ethanol-1% Tween-80 before experiments got an increased transdermal rate, whereas, those only accepted the treatment of 1% Tween-80 showed a decreased transdermal rate. The steady-state infiltration rate of imperatorin had a relationship with drug concentration, with the concentration increase, a bigger steady-state infiltration rate was obtained. When the drug concentration reached a certain concentration, the value was no longer changed. From the above research it could be found that ethanol could change the structure of the skin stratum corneum, which resulted in the increase of steady-state infiltration rate. The effect of Tween-80 on the steady-state permeation rate had nothing to do with skin structure. The concentration of drug in the diffusing cell had an effect on the penetration of imperatorin, which was the combined action of different diffusion route.