The present study investigates the efficacy of polyethylenimine (PEI)-DNA complex that expressed human telomerase reverse transcriptase (hTERT) to transfect hair follicle stem cells and produce sufficient hTERT to stimulate hair growth. Transfection with pLC-hTERT-DNA-PEI complex (D+P group) in vitro induced expression of proliferating cell nuclear antigen in 35.8% of the purified stem cell population, suggesting enhanced cell proliferation. In vivo transfection efficiency of rat dorsal skin was determined by staining for β-gal activity. Cells positive for β-gal were located in the bulge region and dermal sheath of hair follicles. The follicles in the hTERT-transfected region entered anagenon day 15 after transfection, whereas non-transfected (Neg) controls remained in telogen. The similar effect was observed in 50-day-old rat dorsal skin. D+P group displayed a specific expression of hTERT and sufficient to initiate a transition to the anagen phase and promote new hair synthesis 18 days after the transfection. hTERT promoted follicle neogenesis following wounding. In all, 60 days after wounding, tissues of the D+P group showed more newly regenerating hair follicles (83±52 regenerated follicles per rat) in contrast to control group tissues (15±15 regenerated follicles per rat). These studies provide a potential approach for gene therapy of skin disease.