OBJECTIVE： To study the percutaneous absorption of 0.03％ Tacrolimus ointment， and to compare the difference of domestic test preparation and imported reference preparation. METHODS： Modified Franz diffusion cells were adopted in transdermal test in vitro； HPLC-MS method was used to determine permeation amount and rate in vitro， delay time of domestic test preparation and imported reference preparation 0.03％ Tacrolimus ointment. RESULTS： 24 h in vitro permeation amount of test and reference preparations were （3 907±1 191） and （3 896±1 064） ng/cm2； permeation rates were 186.7 and 182.9 ng/（cm2·h）； delay time were 1.95 and 2.00 h， respectively（P＞0.05）. CONCLUSIONS： Test preparation shows good percutaneous property， and is similar to reference preparation in penetration absorbency through nude mice skin.