Objective Dermal papillae cells are widely applied to reconstruction of tissue engineered hair foll icle and skin. To investigate the difference of the biological characteristics of dermal papillae cells cultured with keratinocyte medium (KM)and normal medium (NM), and to determin whether it is feasible for the reconstruction of tissue engineered hair foll icle using dermal papillae cells cultured in KM. Methods Scalp samples were obtained in rhytidectomy procedure. Dermal papillaes were isolated by two steps digestive treatment, then cultured with KM and NM in two groups. The time of dermal papillae adherence and cell outgrowth was recorded and the rate of dermal papillae adherence was determined after 5 days. As well as, the difference of cell morphology was observed through inverted phase contrast microscope. The maximum generations were determined in two groups and the cell sheets were observed by HE staining. In third-generation cells, the number of aggregates in every dish and the prol iferation by MTT were compared between two groups. Meanwhile, the expression of α-smooth muscle actin (α-SMA) and ALP were detected by immunofluorescence and specific staining in two groups. Results Dermal papillaes of KM group had a higher rate of adherence and fast outgrowth. The rates of adherence were 54.17% and 36.78% in KM group and in NM group, respectively. In KM group, cells adhered after 24 hours and outgrew after 64 hours. While, cells adhered after 48 hours and outgrew after 80 hours in NM group. The cells were bigger in NM group than in KM group. In third-generation cells, 3.06 ± 1.12 and 9.25 ± 1.73 aggregates formed in NM group and KM group, respectively, the difference was significant (P lt; 0.05). In addition, cells could form cell sheets which were muti-layers in KM group. Mostly 7 and 15 generations could been subcultured in NMgroup and KM group, respectively. The result of MTT indicated that cells prol iferated more actively in KM group; absorbance value of KM group was significantly higher than that of NM group after 7 days (P lt; 0.05). The positive of α-SMA were detected in the third-generation cells of both groups. Ocassionally a l ittle few cells expressed ALP with (987 ± 146) m2 positive area in the sixth-generation cells of NM group. However, the cells still expressed ALP with (8 757 ± 558) μm2 positive area in the fourteenthgeneration cells of KM group and the difference was significant (P lt; 0.05). Conclusion Cells proliferate actively and aggregate obviously and could been subcultured more generations in KM. Therefore, culturing dermal papillae cells with KM is feasible for the reconstruction of tissue engineered hair foll icle.