Objective To explore an effective method for culturing perineurial cells in vitro, and to preliminarily study the role of hair follicle neural crest stem cells (hfNCSCs) in activating perineurial cells. Methods Perineurial cells from rat sciatic nerve were cultured and purified by the method of "limited digestion-differential adherence-chemical drug", hfNCSCs from rat vibrissa were cultured, and the cells were identified by immunocytochemistry staining. hfNCSCs and perineurial cells were co-cultured in Transwell plates, where perineurial cells were seeded in the upper chamber, and hfNCSCs (hfNCSC co-culture group) or acellular grids (control group) were seeded in the bottom chamber. Crystal violet staining was performed after co-culture for 6, 12 and 18 h to observe the migration of perineurial cells. The perineurial cells were treated with hfNCSCs conditioned medium (hfNCSC conditioned medium group) and 2% FBS DMEM medium (control group), respectively, and the proliferation of perineurial cells was detected by cell counting kit 8 (CCK-8) after 24, 48 and 72 h. Results Perineurial cells with purity up to (97.66±2.08)% were obtained within 2 weeks by this method. The migration number of perineurial cells was significantly higher in the hfNCSC co-culture group than in the control group after 6, 12 and 18 h of co-culture (P<0.05, P<0.01). The cell viability of perineurial cells in the hfNCSC conditioned medium group and control group was similar after treated with hfNCSCs conditioned medium for 24 and 48 h (both P>0.05); however, the cell viability of perineurial cells was significantly higher in the hfNCSC conditioned medium group than in the control group after 72 h (P<0.01). Conclusion Perineurial cells can be successfully cultured and purified by the method of "limited digestion-differential adherence-chemical drug"; hfNCSCs can activate perineurial cells and promote their migration and proliferation.