Background: AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that plays a pivotal regulator in the balancing cellular energy metabolism. Recent studies have been reported that AMPK has numerous roles in physiological conditions and dysregulation of AMPK induces pathological processes and serious diseases. However, AMPK and its activators have not been studied to determine whether they regulate hair growth. Methods: To investigate the role of AMPK pathway in hair growth, we screened metformin, a well-known AMPK activator, to determine whether they have the beneficial effect of hair growth. In addition, to evaluate the molecular and cellular mechanisms, protein levels of AMPK and β-catenin were analyzed. Results: Metformin increased the cellular proliferation in human dermal papilla cell (DP) and outer root sheath cell (ORS). Ki-67 expression also significantly increased with metformin treatment in the ex vivo hair follicle organ culture. Moreover, metformin affected the anagen phase by maintaining interaction between hair matrix and DP cells. In further study to investigate the mechanism by which AMPK regulates hair growth, treating DP and ORS cells with metformin led to the significant increase of AMPK phosphorylation, which in turn suppressed β-catenin degradation and enhanced its nuclear accumulation. Conclusion: We demonstrated that metformin promotes hair growth via AMPK/β-catenin signaling pathway in vitro, affecting in DP and ORS cells. The hair promoting effects of AMPK activators might be a new proposal in treatment of alopecia, and further investigation will be needed in the future.