Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by cutaneous lesions and intense pruritus. The warm temperature-activated Ca2+-permeable TRPV3 channel is abundantly expressed in the keratinocytes, and gain-of-function mutations of TRPV3 cause skin lesions and pruritus in rodents and humans, suggesting an involvement of TRPV3 in the pathogenesis of AD. Here we report that pharmacological and genetic inhibition of TRPV3 attenuates skin lesions and dermatitis in mice. In mouse AD-like model induced by topical application of chemical DNFB, we found that TRPV3 proteins together with inflammatory factors TNF-α and IL-6 were upregulated in the skin detected by Western blot and immunostaining assay. Pharmacological activation of TRPV3 by channel agonist skin sensitizer carvacrol resulted in development of AD in WT mice, but not TRPV3 knockout mice. Furthermore, inhibition of TRPV3 by natural osthole reversed the severity of inflammatory dorsal skin and ear edema in dose-dependent manner, and also decreased the expression of inflammatory factors TNF-α and IL-6. Taken together, our findings demonstrate the involvement of overactive TRPV3 in the progressive pathology of AD in mice, and topical inhibition of TRPV3 channel function may represent an effective prevention and therapy for AD or inflammatory skin diseases. SIGNIFICANCE STATEMENT: Overactive TRPV3 channel is critically involved in the pathogenesis of atopic dermatitis. Inhibition of TRPV3 channel function by topical natural osthole may represent an effective therapy for management of atopic dermatitis aimed at preventing or alleviating skin lesions and severe itching.