Atopic dermatitis (AD) is often concomitant with increased levels of IgE against not only foreign allergens but also autoallergens. AD patients with autoallergy are likely to be more severe and difficult to treat, and self-reactive IgE might be a contributing factor in the pathogenesis of AD. However, how autoallergens are recognized by the immune system and what immune responses are induced subsequently remain largely unknown. We found that the serum level of IgE against transglutaminase 3 (TGase3) was significantly higher in AD patients than in healthy individuals and was positively correlated with disease severity. The expression of TGase3 in the lesional skin of AD patients was markedly increased compared with that of the controls, and Th2 cytokines and/or allergen promoted the expression of TGase3 in keratinocytes. TGase3 bond monocytes-derived dendritic cells (MoDCs) via dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), which resulted in the production of IL-6 and activation of the NF-κB signaling pathway in MoDCs; and TGase3-treated MoDCs facilitated Th1 polarization. Moreover, skin inflammation in the mouse model of MC903-induced AD was attenuated when TGase3 was inhibited. In conclusion, TGase3 was revealed as an autoallergen in AD and actively involved in skin inflammation; TGase3-targeting might be a therapeutic strategy for the treatment of AD.