Gintonin-enriched fraction (GEF), a bioactive glycolipoprotein derived from <i>Panax ginseng</i> is known for its potential as a natural anti-inflammatory agent. Keratinocytes are closely related to the development and progression of various inflammatory skin conditions. However, the effect of GEF on inflammation-related responses in keratinocytes remains unclear. This study aimed to investigate whether GEF modulates key inflammatory responses in keratinocytes stimulated by tumor necrosis factor (TNF)-α. The effect of GEF on biological activities in TNF-α-stimulated keratinocytes (HaCaT cells) was evaluated using water-soluble tetrazolium salt, enzyme-linked immunosorbent, immunostaining, and immunoblotting assays. In TNF-α-stimulated HaCaT cells, GEF attenuated reactive oxygen species production, nitric oxide release, and inducible nitric oxide synthase expression. Moreover, GEF reduced the release of interleukin (IL)-6 and RANTES, while increasing the release of IL-10 in TNF-α-exposed HaCaT cells. Additionally, GEF treatment resulted in reduced cyclooxygenase-2 expression and prostaglandin E₂ release and inhibited TNF-α-induced translocation of nuclear factor-κB in HaCaT cells. Furthermore, TNF-α and IL-6 levels in ultraviolet B-irradiated HaCaT cells were reduced by GEF treatment. These findings indicated that GEF exerts anti-inflammatory effects on keratinocytes. This study provides a basis for the development of novel therapeutic approaches for the prevention and treatment of inflammatory skin disorders.