The dermal papilla (DP) plays pivotal roles in hair follicle morphogenesis and cycling. In the present study, we attempted to indentify the genes specifically upregulated in freshly microdissected, non-cultured human DP, develop the optimal culture condition that maintains the expression levels of those DP intrinsic genes, and regenerate human DP equivalent with hair inductive activity, using DP cells cultured in this newly established condition. Microarray comparison among freshly isolated DP, conventionally cultured DP cells and fibroblasts enabled the identification of the genes overrepresented in intact human DP (DP genes), which potentially contribute to the biological distinctiveness of DP. Interestingly, many of newly identified DP genes were involved in organ development, cell communication and signaling pathways, especially, WNT, BMP and FGF pathways. Next, the optimization of DP culture condition was performed by the supplementation of WNT, BMP and FGF activators into the conventional condition. Those factors were combined at various concentration/ratios and added to DP culture. Finally, the condition that best reproduced DP gene expression profile was identified. In this condition, the expression levels of classic DP genes, including ALPL, WNT5A and NOG, as well as novel DP genes represented by RGS2, were maintained over passages. Intriguingly, re-aggregation of optimally cultured DP cells further ameliorated ALPL, WNT5A and BMP4 expression levels, comparable to that of freshly-isolated human DP. Furthermore, those DP cell aggregates could induce hair follicle-like structures in in vivo hair reconstitution assay. The list of human DP genes and the DP regeneration protocol developed in this study could not only enhance our understanding of the molecular mechanism that underlies hair inductive capacity of DP, but also provide valuable tools for the tissue engineering of human hair follicles.