The study elucidated the wound healing and hair regeneration properties of a conditioned medium prepared from the culture of human hair follicle mesenchymal stem cells (HFMSCs). The wound-healing effects of mesenchymal stem cell-conditioned medium (MSC-CM) were tested in vitro using scratch assays co-cultured with HaCaT keratinocyte and monitored through optical microscopy. The cell proliferation of HFMSCs and the HaCaT keratinocyte were observed in the presence of different kinds of drugs including UK5099, sodium L-lactate, lactate dehydrogenase-A, MSC-CM, caffeine, and caffeic acid. The hair regeneration properties were investigated in vivo by administrating the MSC-CM solutions to adult B6 mouse models. For quantification, hematoxylin and eosin staining were performed following euthanasia. In vitro results revealed that MSC-CM promotes dermal cell migrations and enhances proliferation of HFMSCs and HaCaT keratinocytes, demonstrating wound-healing properties. Moreover, when the MSC-CM solutions were applied to the shaved mouse skin, a dark area that expanded overtime was seen. Although no hair growth was found, histological analysis proved that a fat layer thickness increment was found under the mouse’s skin, ultimately projecting the formation of new hair growth. MSC-CM promotes the migration and proliferation of dermal keratinocytes that are beneficial for wound healing and hair growth. It is believed that MSC-CM can potentially serve as the basis of alternative therapeutic applications for wound closure and skin regeneration as well as hair growth stimulation and hair loss prevention in alopecia.