Trypsin has been extensively used in laboratory settings for in vitro epidermal separation and keratinocyte isolation for over 50 years. The aim of this study was to assess the enhancing effect of trypsin on the transdermal delivery of insulin by applying its specific biochemical properties to react with the stratum corneum (SC) of skin. Bovine insulin was used as a model peptide to investigate in vitro permeation through rat skin and in vivo hypoglycemic effects of bovine insulin with or without the trypsin pretreatment. Trypsin significantly increased the transdermal permeability of bovine insulin in pH 3.0 solution, but no effect was observed in pH 6.0 solution. The permeation flux of bovine insulin from pH 3.0 solution was promoted 5.2-fold with 0.25% trypsin pretreatment when compared with the control. The enhancement of trypsin was dependent on the concentration in the range of 0.5-2.5%. Furthermore, with trypsin pretreatment, the plasma glucose level was reduced to less than 60% of the initial value after 8 h of in vivo permeation of bovine insulin with pH 3.0 solution, but did not return to the initial value during an 8-h experiment. Mechanistic studies with Fourier transform-infrared and attenuated total reflectance analysis and electrical resistance measurements suggest that trypsin alters the SC protein structure from the alpha- to the beta-form and decreases the electrical resistance of skin, thereby decreasing the SC barrier and enhancing the permeation of insulin. We conclude that trypsin would be effective as a biochemical enhancer for the transdermal delivery of peptide and protein drugs such as insulin.