This study investigated the role of CD226 in a 2,4-dinitrochlorobenzene (DNCB)–induced mouse model of atopic dermatitis. The results showed that the lack of CD226 (global and CD4+ T-cell specific) significantly increased ear thickness, reddening, swelling, and scaling of the skin as well as inflammatory cell and mast cell infiltration. RT-qPCR results demonstrated that the mRNA expressions of atopic dermatitis–related inflammatory cytokines and chemokines were markedly increased in the draining lymph nodes and lesioned ear skin tissues of global and CD4+ T-cell–specific CD226-deficient mice compared with that in control mice. In vitro assessment revealed that CD226 directly modulates TGFβ-mediated regulatory T (Treg) cell differentiation and proliferation. Notably, Treg cell–specific deletion of CD226 (Cd226fl/flFoxp3cre mice) resulted in more severe dermatitis and epidermal thickening than those observed in littermate mice upon DNCB treatment. Subsequent analysis showed that the infiltration of Treg cells in ear lesions and the number of Tregs in the spleen were significantly reduced in Cd226fl/flFoxp3cre mice after DNCB treatment. In addition, the lack of CD226 induced apoptosis of Treg cells through the activation of caspase 3. Therefore, these results suggest that CD226 has potential efficacy in atopic dermatitis, correlating with Treg cell inhibition.