The influence of temperature on the transdermal permeation enhancing mechanism of borneol (BO) was investigated using a multi-scale method, containing a coarse-grained molecule dynamic (CG-MD) simulation, an in vitro permeation experiment, and transmission electron microscope (TEM) study.The results show that BO has the potential to be used as a transdermal penetration enhancer to help osthole (OST) penetrate into the bilayer.With the increasing temperature, the stratum corneum (SC) becomes more flexible, proving to be synergistic with the permeation enhancement of BO, and the lag time (Tlag) of BO and OST are shortened.But when the temperature increased too much, withthe effect of BO, the structure of SC was destroyed;for example, a water pore was formed and micelle reversed.Though there were a number of drugs coming into the SC, the normal bilayer structure was absent.In addition, through comparing the simulation, in vitro experiment, and TEM study, we concluded that the computer simulation provided some visually detailed information, and the method plays an important role in related studies of permeation.