Background: Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by severe itching and recurrent eczema-like lesions. Yet, its exact pathological mechanism remains unclear. Objective: The aim of this study was to investigate the role of TRPA1 in the pathogenesis of AD. Methods: The experimental atopic dermatitis (AD)-like skin lesions were established using 2,4-dinitrochlorobenzene (DNCB). Mice were divided into three groups: TRPA1−/− and WT groups were treated with DNCB dissolved in a 3:1 mixture of acetone and olive oil and the negative control group was treated with 3:1 mixture of acetone and olive oil without DNCB. The treatment lasted for 21 days, after which the animals were sacrificed and their blood, ears and dorsal skin tissue samples were collected for analysis. Results: Lower dermatitis score, ear thickness, pruritus score, and epidermal hyperplasia were observed in mice in TRPA1−/− mice compared to the WT group. Besides, lower dermal mast cell infiltration, proinflammatory cytokines, Th2 cytokines and the infiltration of macrophages were observed in the TRPA1−/− mice compared to the WT group. Furthermore, we demonstrated that TRPA1 antagonist HC-030031 could alleviate AD-like symptoms and reduce the degree of epidermal hyperplasia in mice. Conclusions: TRPA1 has a crucial role during the AD pathogenesis in mice, thus could be used as a potential new target for treating patients with chronic skin inflammatory disease.