Scope: Extra virgin olive oil (EVOO) is rich in phenolic compounds, including hydroxytyrosol (HTy) and hydroxytyrosyl acetate (HTy-Ac), which have presented multiple beneficial properties. Their impact on inflammatory responses in human keratinocytes and modes of action have not been addressed yet. Methods and results: Primary human keratinocytes are pretreated with HTy-Ac or HTy for 30 min and stimulated with IL-1 or Toll-like receptor 3 ligand (TLR3-l). Thymic stromal lymphopoietin (TSLP), measured by ELISA, is attenuated by both polyphenols in a dose-dependent manner. The expression of several inflammation-related genes, including distinct TSLP isoforms and IL-8, are assessed by quantitative RT-PCR and likewise inhibited by HTy-Ac/HTy. Mechanistically, EVOO phenols counteracts I B degradation and translocation of NF-B to the nucleus, a transcription factor of essential significance to TSLP and IL-8 transcriptional activity; this is evidenced by immunoblotting. Accordingly, NF-B recruitment to critical binding sites in the TSLP and IL-8 promoter is impeded in the presence of HTy-Ac/HTy, as demonstrated by chromatin immunoprecipitation. Promoter reporter assays finally reveal that the neutralizing effect on NF-B induction has functional consequences, resulting in reduced NF-B-directed transcription.