The aryl hydrocarbon receptor (AhR) is known to bind several exogenous ligands such as natural plant flavonoids, synthetic polycyclic aromatic hydrocarbons, and dioxin-like compounds, but its association with kaempferol (KF) is unknown. Therefore, in this study, AhR was explored as an atopic dermatitis (AD)-regulating target of KF for AD regulation using in silico prediction, and pharmacological prediction and various AD-induced models confirmed that KF exhibited pharmacological activity through AhR regulation. Also, the results of the study showed that KF regulated epidermal differentiation terminal proteins through the AhR pathway in HaCaT cells stimulated with TNF-α/IFN-γ, and the therapeutic effect of KF was also proven in an AD-induced mouse model and a reconstructed human skin model. In this study, AhR was explored as a KF–AD combined treatment target through in silico prediction analysis, and KF was proven to have an AD treatment effect through AhR regulation in vitro and vivo and in the reconstructed human skin model. In particular, KF can be used as a potent inducer of AhR signaling because it protects against AD by enhancing epidermal terminal differentiation through the AhR-mediated pathway in keratinocytes.