Atopic dermatitis (AD) is a globally increasing chronic inflammatory skin disease with limited and potentially side-effect-prone treatment options. Monotropein is the predominant iridoid glycoside in Morinda officinalis How roots, which has previously shown promise in alleviating AD symptoms. This study aimed to systematically investigate the pharmacological effects of monotropein on AD using a 2, 4-dinitrochlorobenzene (DNCB)/Dermatophagoides farinae extract (DFE)-induced AD mice and tumor necrosis factor (TNF)-α/interferon (IFN)-γ-stimulated keratinocytes. Oral administration of monotropein demonstrated a significant reduction in AD phenotypes, including scaling, erythema, and increased skin thickness in AD-induced mice. Histological analysis revealed a marked decrease in immune cell infiltration in skin lesions. Additionally, monotropein effectively downregulated inflammatory markers, encompassing pro-inflammatory cytokines, T helper (Th)1 and Th2 cytokines, and pro-inflammatory chemokines in skin tissues. Notably, monotropein also led to a considerable decrease in serum immunoglobulin (Ig)E and IgG2a levels. At a mechanistic level, monotropein exerted its anti-inflammatory effects by suppressing the phosphorylation of Janus kinase / signal transducer and activator of transcription proteins in both skin tissues of AD-induced mice and TNF-α/IFN-γ-stimulated keratinocytes. In conclusion, monotropein exhibited a pronounced alleviation of AD symptoms in the experimental models used. These findings underscore the potential application of monotropein as a therapeutic agent in the context of AD, providing a scientific basis for further exploration and development.