The transdermal permeation and metabolic characteristics of methyl nicotinate (MN) in stratum corneum and split-thickness human skin and three species of shed snake and snake skin (Elaphae obsoleta, Naja kaouthia, and Python molurus bivittatus) were evaluated. In vitro skin transport using excised skin and hydrolysis experiments using skin homogenate were carried out. The flux of MN, a metabolite, nicotinic acid (NA), and the total (MN+NA), as well as kinetic parameters (Vmax and Km) for hydrolysis of MN were determined and compared among various skin types. The total flux from MN-saturated solution through human skin was not significantly different from that through snake and shed snake skin of Elaphae obsoleta, Naja kaouthia but was significantly higher than that through snake and shed snake skin of Naja kaouthia (p < 0.05). A great difference in skin esterase activity was observed between human and snake in both snake skin and shed snake skin of all species. In all skins except the stratum corneum of human skin, NA flux increased with an increase in MN donor concentration and reached a plateau, suggesting that metabolic saturation was taking place in the skin. NA flux at the plateau and MN donor concentrations at which the NA flux reached a plateau also varied by species. These findings indicated that the discrepancy in transdermal profiles of MN among skins tested was predominantly due to the difference in the esterase activity in the skin.