Atopic dermatitis(AD) is a chronic inflammatory skin disorder characterized by immune dysregulation and impaired epidermal barrier integrity. Keratinocytes contribute to the persistence of AD by producing inflammatory mediators in response to cytokine stimulation. Inonotus obliquus, commonly known as chaga mushroom, contains bioactive compounds with antioxidant and anti-inflammatory properties. However, the effects of its water extract on inflammatory responses and barrier-associated changes in keratinocytes remain unclear. This study investigated the activity of Inonotus obliquus water extract (IOE) in TNF-α/IFN-γ-stimulated HaCaT keratinocytes, an in vitro model reflecting major features of inflammatory skin conditions. The effects of IOE on inflammatory mediator production, intracellular signaling, and the expression of proteins associated with epidermal barrier function were evaluated. IOE attenuated cytokine-induced inflammatory responses by regulating major inflammation-related signaling pathways. It also alleviated inflammation-associated changes in barrier-related protein expression, indicating a possible protective effect on keratinocyte function. These findings suggest that IOE can attenuate inflammatory responses and partially restore barrier-related protein expression under inflammatory conditions. IOE may therefore be a promising natural material for further investigation as a supportive approach for AD and related inflammatory skin disorders.