Considerable attention has focused on the health-promoting effects of red wine and its nonflavonoid polyphenol compound resveratrol. However, the underlying molecular mechanisms and molecular target(s) of red wine or other potentially active ingredients in red wine remain unknown. Here, we report that red wine extract (RWE) or the red wine flavonoid quercetin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation of JB6 promotion-sensitive mouse skin epidermal (JB6 P+) cells. The activation of activator protein-1 and nuclear factor-kappaB induced by TPA was dose dependently inhibited by RWE or quercetin treatment. Western blot and kinase assay data revealed that RWE or quercetin inhibited mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) 1 and Raf1 kinase activities and subsequently attenuated TPA-induced phosphorylation of ERK/p90 ribosomal S6 kinase. Although either RWE or quercetin suppressed Raf1 kinase activity, they were more effective in inhibiting MEK1 activity. Importantly, quercetin exerted stronger inhibitory effects than PD098059, a well-known pharmacologic inhibitor of MEK. Resveratrol did not affect either MEK1 or Raf1 kinase activity. Pull-down assays revealed that RWE or quercetin (but not resveratrol) bound with either MEK1 or Raf1. RWE or quercetin also dose dependently suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are involved in the activation of MEK/ERK signaling. Docking data suggested that quercetin, but not resveratrol, formed a hydrogen bond with the backbone amide group of Ser(212), which is the key interaction for stabilizing the inactive conformation of the activation loop of MEK1.