Itch in atopic dermatitis (AD) is aggravated under warm conditions. Transient receptor potential vanilloid 3 (TRPV3), a member of the thermosensitive TRP channels, is activated by innocuous heat and is abundantly expressed in keratinocytes. The potential role of TRPV3 in itch is illustrated in TRPV3 channelopathies of human and mice. However, the role of TRPV3 in heat-induced itch in AD and the underlying mechanisms are unclear. Here we showed that keratinocytes isolated from patients with AD exhibit enhanced expression and heat sensitivity with hyperactive channel function of TRPV3. Heat stimulus induced enhanced secretion of thymic stromal lymphopoietin (TSLP), nerve growth factor (NGF), and prostaglandin E2 (PGE2) by keratinocytes from AD patients via TRPV3 activation. TRPV3 agonists increased TSLP, NGF, PGE2, and IL-33 production in human keratinocytes and induced scratching behavior upon intradermal injection in mice. TRPV3 was upregulated in the skin of MC903-induced AD mouse model. Heat stimulation to MC903-treated mice increased scratching behavior and produced higher levels of TSLP, NGF, PGE2, and IL-33 from epidermis, which were attenuated by pharmacological inhibition of TRPV3. Moreover, neutralization of TSLP reduced heat-evoked scratching in MC903-challenged mice. These results suggest that TRPV3 is a potential therapeutic target for heat-induced itch in AD.