5-Hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF), a polymethoxyflavone, is found exclusively in the Citrus genus, particularly in the peels of sweet orange. Herein, we report the first investigation of the inhibitory effects of 5-OH-HxMF on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in mouse skin. We found that the topical application of 5-OH-HxMF can effectively inhibit the transcriptional activation of iNOS and COX-2 mRNA and protein in mouse skin stimulated by TPA. Pre-treatment with 5-OH-HxMF resulted in the reduction of TPA-induced nuclear translocation of nuclear factor-kappaB (NF-kappaB) subunit and DNA binding by blocking phosphorylation of inhibitor kappaB (IkappaB) alpha and p65 and subsequent degradation of IkappaBalpha. In addition, 5-OH-HxMF can inhibit TPA-induced phosphorylation and nuclear translocation of the signal transducer and activator of transcription-3. Moreover, 5-OH-HxMF can suppress TPA-induced activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt, which are upstream of NF-kappaB. We also found that 5-OH-HxMF significantly inhibited TPA-induced mouse skin inflammation by decreasing inflammatory parameters. Furthermore, 5-OH-HxMF significantly inhibited 7,12-dimethylbenz[a]anthracene/TPA-induced skin tumor formation by reducing the tumor incidence and tumor multiplicity of papillomas at 20 weeks. Therefore, all these results revealed for the first time that 5-OH-HxMF is an effective antitumor agent and its inhibitory effect is through the down-regulation of inflammatory iNOS and COX-2 gene expression in mouse skin, suggesting that 5-OH-HxMF is a novel functional agent capable of preventing inflammation-associated tumorigenesis.