Polyozellus multiplex is an edible mushroom that offers beneficial pharmacological effects against intestinal inflammation and cancer. Previous studies have demonstrated that polyozellin, a major component of P. multiplex, has therapeutic activities against inflammation, cancer, and oxidative stress-related disorders. This study aimed to determine the pharmacological effects of polyozellin on inflammatory and pruritic responses, the major symptoms of atopic dermatitis (AD), and to define its underlying mechanism of action. Our results showed that polyozellin inhibited the expression of inflammatory cytokines and chemokines through blockade of signal transducer and activator of transcription 1 and nuclear factor-κB in activated keratinocytes, the major cells involved in AD progression. Based on the histological and immunological analyses, oral treatment with polyozellin attenuated the Dermatophagoides farinae extract (DFE)/2,4-dinitrochlorobenzene (DNCB)-induced atopic inflammatory symptoms in the skin. Pruritus is an unpleasant sensation for AD patients that causes scratching behavior and ultimately exacerbates the severity of AD. To find a possible explanation for the anti-pruritic effects of polyozellin, we investigated its effects on mast cells and mast cell-derived histamines. Oral treatment with polyozellin reduced the DFE/DNCB-induced tissue infiltration of mast cells, the serum histamine levels, and the histaminergic scratching behaviors. Additionally, polyozellin decreased the immunoglobulin E-stimulated degranulation of mast cells. Taken together, the findings of this study provide us with novel insights into the potential pharmacological targets of polyozellin for treating AD by inhibiting the inflammatory and pruritic responses.