AbstractReconstruction of skin equivalents with physiologically relevant cellular and matrix architecture is indispensable for basic research and industrial applications. As skin-nerve crosstalk is increasingly recognized as a major element of skin physiological pathology, the development of reliable in vitro models to evaluate the selective communication between epidermal keratinocytes and sensory neurons is being demanded. In this study, we present a three-dimensional innervated epidermal keratinocyte layer as a sensory neuron-epidermal keratinocyte co-culture model on a microfluidic chip using the slope-based air-liquid interfacing culture and spatial compartmentalization. Our co-culture model recapitulates a more organized basal-suprabasal stratification, enhanced barrier function, and physiologically relevant anatomical innervation and demonstrated the feasibility of in situ imaging and functional analysis in a cell-type-specific manner, thereby improving the structural and functional limitations of previous coculture models. This system has the potential as an improved surrogate model and platform for biomedical and pharmaceutical research.