The development of a simple, easy-to-use, and noninvasive vaccination system is in high demand. For transcutaneous immunization (TCI), we previously reported that a hydrogel patch was an effective TCI device that accelerates antigen penetration through the stratum corneum in mouse and rat models. The present study was performed to characterize the transcutaneous protein delivery induced by the hydrogel patch in mouse, guinea pig, LWD pig, human, or tissue-engineered skin models, and to assess the activity of proteins delivered into the skin. The hydrogel patch promoted protein penetration through the stratum corneum in all skin models, indicating that our original hydrogel patch might have practical application for use in humans. In addition, proteins delivered into the skin by the hydrogel patch retained their activity, suggesting that the hydrogel patch is applicable for the delivery of therapies for other diseases as well. On the basis of these results, translational research in human is now in progress.