Transient receptor potential vanilloid 4 (TRPV4) is a Ca²⁺-permeable non-selective cation channel and its activating stimuli include anandamide, bisandrographolide, citric acid, arachidonic acid metabolic products by epoxygenases, hypo-osmotic cell swelling, and warm temperature. TRPV4 is involved in Ca²⁺-dependent signal transduction in several tissues. Since the activation of TRPV4 facilitates adherence-junction formation in the skin epithelium, compounds that activate TRPV4 are expected to maintain or improve the barrier function of epidermal cells. In this study, we found that the extract of Arachis hypogaea (A. hypogaea) activates human TRPV4 (hTRPV4). In the Ca²⁺-imaging experiment, the application of A. hypogaea extract exhibited the intracellular Ca²⁺ concentration ([Ca²⁺]_i) increases in HEK293T cells expressing hTRPV4. The [Ca²⁺]_i increases by application of A. hypogaea extract were not observed in HEK293T cells expressing TRPV1, TRPV2, TRPV3, TRPM8, or TRPA1. Moreover, the application of A. hypogaea extract enhanced transepithelial electrical resistance in the keratinocyte monolayer. These results suggest that A. hypogaea extract is useful for maintaining and improving the barrier function of epidermal cells.