Reconstruction of full-thickness skin equivalents with physiologically relevant cellular and matrix architecture is gaining importance as an in vitro tool for basic research, and for the pharmaceutical, toxicological, and cosmetic industries. However, human skin equivalents reconstructed on traditional culture systems are limited by a weak skin barrier function compared to normal human skin. Probable reasons include the lack of mechanical forces and dynamic flow system that provide necessary mechanistic signals and continuous supply and/or drainage of nutrients and metabolites. Here, we combine a fibrin-based dermal matrix with a biomimetic ‘organ-on-chip’ system for the development of human skin equivalents that better recapitulate the structure and functionalities of human skin, compared to conventional static culture systems. We demonstrate that dynamic perfusion and a fine control of the microenvironment enable improved epidermal morphogenesis and differentiation, and enhanced barrier function. It is also shown that integrated 3D culturing and integrity/permeability testing can be conducted directly on the organ-on-chip device owing to the non-contracting properties of the fibrin-based dermal matrix, thus overcoming the limitations of collagen-based skin equivalents used in conventional cell culture inserts and diffusion cells. With this scalable system, it is possible to achieve higher throughput and automation of culture and testing protocols, and deliver low-cost alternatives to animal and clinical studies for drug screening and toxicological applications.