The upper papillary and deeper reticular dermis differ structurally and functionally. Although the papillary and reticular fibroblasts produced distinct extracellular matrix (ECM), the matrisome of these two fibroblast subpopulations has not been defined. Therefore we performed a transcriptomic analysis of papillary and reticular fibroblasts freshly isolated from skin of young donors around 20, at a time they produced high level of ECM. Bioinformatics analysis delivered 230 upregulated and 139 downregulated transcripts in papillary vs reticular fibroblasts. Expression of various selected genes was validated by q-PCR. The papillary fibroblasts were characterized by a higher expression of genes involved in the defense function, in the regulation of cell motility and proliferation and in the MAPK cascade whereas reticular fibroblasts showed higher expression of genes related to the development of connective tissues. Papillary fibroblasts were characterized by the expression of a high number of matrisome-associated genes whereas reticular fibroblasts gene signature mainly related to the core matrisome and ECM regulators. The regulation of selected genes was validated at protein level attesting to the robustness of the transcriptome analysis. Altogether, our data brought new insights into an ECM signature that is coherent with the organization and function of the papillary and reticular dermis.