To study the transdermal diffusion of berberine hydrochloride in Shanghuang Cream with two different Azone contents on different skins and to compare and analyze the effect. Methods: Intelligent transdermal test instrument was used to determine the cumulative permeating amounts of berberine hydrochloride in two kinds of Shanghuang Cream with 1% azone（Cream A）and 4% azone（Cream B）. Then diffusion rate was calculated. Results: The cumulative permeating amounts and transdermal diffusion rate on mice skin, including skin of back and abdomen, were significantly higher than those on snake slough（P<0.01）. On snake slough, the cumulative permeating amounts and transdermal diffusion rate of berberine hydrochloride in cream A was significantly higher than cream B（P=0.037,0.028）. The difference of the cumulative permeating amounts between two creams was not significant on mice skin, but the transdermal diffusion rate of cream B was significantly higher than that of cream A on mice skin of back（P=0.046）. Conclusion: Different skin shows different result. Snake slough was superior to mice skin in optimizing Azone content because of its high sensitivity, less interferences and excellent stability.