Skin is the first protecting barrier of our body from external harmful and maintenance of skin homeostasis, auto-regeneration and wound healing are notably insured by stem cells. While no skin stem cell markers have been clearly identified in human skin, several sub-populations of cells including stem cells, progenitor cells and others have been revealed to be located to the basal layer of epidermis, a stratified epithelium composed of different layers of differentiated and undifferentiated cells. Cells of basal layers are found on the dermo-epithelium junction (DEJ) that presents undulations along skin tissue. Recent studies have shown that a specific subpopulation of cells located on dermal papillae express some proteins that are not or less expressed compared to cells located in rete-ridges. During ageing, a decrease of skin renewing concomitant with a flattening of DEJ is observed. In parallel, the expression of these specific proteins by cells on dermal papillae also decrease during ageing. Thus, these sub-populations of cells in dermal papillae have been classified as interfollicular stem cells and would be required for epidermis homeostasis. Specific physiological environments (« niche ») have been shown as crucial to the maintenance and fate control of specific cell types. By atomic force microscopy, we show here that these different sub-populations of cells present specific mechanical properties depending on cell locations along the DEJ. Indeed, these interfollicular stem cells located on dermal papillae and expressing specific pool of proteins harbour a stiffness profile different from stiffness profile of rete-ridge cells. In addition, we also show that stiffness of these cells decreases during ageing. This is in agreement with a decrease in the maintenance in the pool of interfollicular stem cells during ageing where epidermis homeostasis is altered. Thus, these specific mechanical signatures define new ways to discriminate different sub-populations of cells on the basal layer. Moreover, these specific environments could allow for the maintenance of specific cell pools involved in skin renewing. These results bring new insights about cell fate and cell maintenance, both involved in epidermis renewing. At short and long term, these data could help to improve anti-ageing agent formulation for cosmetic field as well as to ensure quality or integrity of both skin grafts and 3D reconstruction of skin models.