Abstract: To explore the role of autophagy in hair follicle cycle and whether 3-methyladenine(3-MA)could promote hair regeneration in C57BL/6 mice through inhibiting autophagy flux, hair regeneration model of C57BL/6 mice was induced on the dorsal skin by depilation, and 3-MA was intraperitoneally injected to investigate hair regrowth, meanwhile vehicle and rapamycin(RAPA)were used as the controls. Results showed that 3-MA could obviously promote hair regrowth in depilated C57BL/6 mice. Furtherly, haematoxylin-eosin staining, immunohistochemical staining, immunofluorescence and Western blot tests were used to investigate the autophagy signals and the cell proliferation. Results showed that the expression of Beclin1 and LC3B II/I ratio were significantly decreased. Expression of P62 and Ki67 were increased, as well as the CD34 and CD49f double-labeled hair follicle stem cells were obviously increased inside bulge areas in 3-MA group, while contrarily in RAPA group. These results affirmed 3-MA, an autophagy inhibitor, could promote hair regeneration in depilated C57BL/6 mice by facilitating the transformation of hair follicle from telogen to anagen. 3-MA and other analogous autophagy inhibitors probably have a potential usage in future therapy in human telogen effluvium diseases.