Abstract:  Keratinocytes constitute the first‐line barrier against exogenous antigens and contain Toll‐like receptors (TLRs), which function as pattern‐recognition molecules to activate antimicrobial innate immune responses. In an effort to ascertain whether or not filaggrin (filament‐aggregating protein) expression affected the TLR‐mediated responses of keratinocytes, we transfected filaggrin siRNA into HaCaT human keratinocyte cells and determined that thymic stromal lymphopoietin (TSLP) and IL‐6 secretion were increased by poly(I:C) stimulus. Additionally, TSLP expression is increased in filaggrin knockdown as well as TLR3 stimulation in reconstituted human epidermal layers. Therefore, the findings of this study show that reduced filaggrin levels may influence innate immune responses via TLR stimuli and may contribute to the pathogenesis of inflammatory skin disease via TSLP expression.