Alopecia is an exceedingly prevalent problem and lacks effective therapy. Recently, research has focused on early-passage dermal papilla cells (DPCs), which have hair inducing activity both in vivo and in vitro. Our previous study indicated that factors secreted from early-passage DPCs contribute to hair follicle (HF) regeneration. To identify which factors are responsible for HF regeneration and why late-passage DPCs lose this potential，we collected 48-h-culture medium (CM) from both of passage 3 and 9 DPCs and subcutaneously injected the DPC-CM into NU/NU mice. Passage 3 DPC-CM induced HF regeneration, based on the emergence of a white hair coat, but passage 9 DPC-CM not. In order to identify the key factors responsible for hair inductive capacity, CM from passage 3 and 9 DPCs was analyzed by iTRAQ-based quantitative proteomic technology. We identified 1360 proteins, of which 213 proteins were differentially expressed between CM from early-passage vs. late-passage DPCs, including SDF1, MMP3, biglycan and LTBP1.Further analysis indicated that the differentially-expressed proteins regulated the Wnt, TGF-β and BMP signaling pathways, which directly and indirectly participate in HF morphogenesis and regeneration. Subsequently, we selected 19 proteins for further verification by multiple reaction monitoring (MRM) between the two types of CM. These results would be used to identify the key factors for inducing HF regeneration and reveal the molecular mechanisms of losing inductive ability of DPCs. Furthermore, it is possible to explore some drugs for alopecia in the clinic according to this secretome date analysis.