Pleurotus eryngii (also known as king trumpet mushroom, french horn mushroom, king oyster mushroom) is an edible mushroom native to Mediterranean regions of Europe, the Middle East, and North Africa, but also grown in parts of Asia. It has the ability to produce various biologically active compounds and possesses a well-developed ligninolytic enzyme system that participates in the degradation of lignin and different aromatic compounds. In this study, we investigated the protective effects of the ethyl acetate extract of Pleurotus eryngii (PEE) on the development of atopic dermatitis (AD) in human keratinocyte HaCaT cells. Keratinocytes, one of major cell types in the skin, can be induced by TNF-α and IFN-γ to express thymus and activation-regulated chemokine (TARC/CCL17), which is considered to be a pivotal mediator in the inflammatory responses during the development of inflammatory skin diseases, such as AD. In addition, normal T-cell–expressed and secreted chemokine (RANTES) is a (C-C) chemokine released by T lymphocytes, other inflammatory cells, and platelets and plays an important role in allergic inflammatory processes. Pretreatment of HaCaT cells with PEE suppressed TNF-α/IFN-γ-induced protein and mRNA expression of CCL17 and RANTES. PEE significantly inhibited TNF-α/IFN-γ-induced NF-κB activation. These results suggest that PEE may exert anti-inflammatory responses by suppressing TNF-α and IFN-γ-induced activation of NF-κB in the keratinocytes and might be a useful tool in therapy of skin inflammatory diseases.