Dermal Papilla Cells (DPCs) are mesenchymal cells residing at the base of the hair follicles. They play an important role in the hair cycle remodeling and hair follicle regeneration. In this study, DPCs were isolated, and cultured from human hair follicles and evaluated the expression of a-SMA, versican by immunocytochemistry, and alkaline phosphatase activity. The human fibroblasts were used as a control to confirm the non-fibroblast DPCs. The results showed that the cells grew from the hair bulb and had fibroblast morphology. These bulb cells strongly expressed alkaline phosphatase activity a-SMA, and versican while the human fibroblast lacked ALP activity and less expression of a-SMA and versican. In conclusion, the protocol is established to isolate and culture DPCs from hair follicle bulbs, and ALP activity is suggested as the major marker for distinguishing DPCs and human fibroblasts.