The formation and maintenance of the skin barrier are controlled by proliferation and differentiation of epidermal keratinocytes, and impairment of skin barrier homeostasis due to abnormal keratinocyte function causes many skin disorders, including atopic dermatitis and psoriasis (1). Recently, it was reported that the transient receptor potential vanilloid 3 (TRPV3) is functionally expressed in human keratinocytes. Activation of TRPV3 increased intracellular calcium signaling, which in turn increased transglutaminase 1, 3 activities as well as the subsequent formation of cornified cell envelopes in the epidermis (2). These studies suggest that TRPV3 activation could be a potential therapeutic target for skin barrier recovery in various dermatological diseases. The leaves of Agimonia pilosa Ledeb (AP), which have traditionally been used in East Asia to treat conditions including sore throat and eczema, have been reported to possess anti-inflammatory and anti-allergic effects in ovalbumin-induced mouse models (3). In this study, we examined the effects of AP extract on the skin barrier formation via modulation of TRPV3 to evaluate for use in topical agents for treatment of skin barrier disorders. Two types of AP extract were prepared by refluxing 200 g of dried AP with either hot water (APH2O) or 30% ethanol (APEtOH) for 3 hours. We assessed whether the AP extracts showed agonistic effects on the TRPV3 channel. 0.1 mg/mL APH2O strongly activated TRPV3 current (ITRPV3), showing 0.91 ± 0.03% activation compared to 2-APB, a potent agonist of TRPV3 (n = 5); hence, APEtOH cannot activate ITRPV3. We then investigated whether APH2O subsequently activated transglutaminase (TGase) in human primary keratinocytes. We found that TGase activity increased with 0.1 mg/mL APH2O treatment (73 ± 3%, n = 3), which was counteracted by treatment with 50 μM ruthenium red (R.R), an inhibitor of TRPV3. Tape stripping of the stratum corneum is usually applied in research to induce epidermal barrier disruption. Therefore, we performed a tape-stripping test to elucidate whether topical APH2O treatment improves epidermal permeability barrier function in murine skin. Transepidermal water loss (TEWL) was measured at 6, 9, 24, and 30 hours after tape stripping of murine ventral skin. Topical application of APH2O accelerated skin barrier recovery at all time points, and a significant change was observed at 30 hours after barrier disruption (10.9 ± 0.24% versus control, n = 3). These results suggest that topical APH2O treatment could be useful for the prevention and treatment of skin barrier disorders.