Skin is a thin, stratified tissue covering the entire body. It is microscopically defined by layers of epidermal epithelial cells separated from cells in the dermis by a basement membrane (BM), the skin’s extracellular matrix (ECM). The skin’s ECM binds the layers together to form the tissue’s organization and plays important roles in regulation of skin morphogenesis and repair. In this study, skin ECM components have been identified and their interactions with cells characterized using large-scale, quantitative proteomic analysis of skin biomatrix scaffolds, decellularized tissue extracts highly enriched in skin ECM. These were used to quantify structure and function of the skin’s matrix, referred to as the Matrisome, comprised of core ECM components (collagens, proteoglycans and glycoproteins) and of ECM-associated factors, matrix-bound, soluble signals that are key regulators of epidermal development. Analyses on protein-protein interactions of ECMs showed a mechanistic link between hemidesmosome assembly and epidermal homeostasis. This was achieved largely through the integrated network of the hemidesmosome complex and associated proteins mediating anchorage of epidermal stem/progenitors onto skin ECM. These results highlight the power of large-scale proteomics to identify the components and functions of the skin Matrisome regulating tissue homeostasis and ECM remodeling.