METHODS Hydroxylated polymethoxyflavones (PMFs), existing exclusively in citrus genus, have been reported to exhibit a broad spectrum of biological activity. Here we investigated the chemopreventive effects and underlying molecular mechanisms of dietary administration of hydroxylated PMFs in an azoxymethane (AOM)-induced colonic tumorigenesis model. RESULTS Male, Institute of Cancer Research (ICR), mice at age of 6 wk were injected with AOM twice weekly at a dose of 5 mg/kg for 2 wk and continuously fed control diet or diets containing 0.01 and 0.05% hydroxylated PMFs, respectively. Mice were then sacrificed at 6 and 20 wk, and colonic tissues were collected and examined. Hydroxylated PMFs feeding dose-dependently decreased the number of aberrant crypt foci in colonic tissues of mice. More importantly, we found that hydroxylated PMFs caused a strong reduction in numbers of large aberrant crypt foci and tumors in colonic tissue. Molecular analysis exhibited the anti-proliferative, anti-inflammatory, anti-angiogenic and pro-apoptotic activities of hydroxylated PMFs by significantly decreasing the levels of inducible nitric oxide synthase, cyclooxygenase, cyclin D1 and vascular endothelial growth factor through interfering with Wnt/β-catenin and epidermal growth factor receptor/Ras/mitogen-activated protein kinase signaling pathways as well as the activation of transcription factors NF-κB and STAT3 in colonic tissue, thus resulting in suppression of colonic tumorigenesis. CONCLUSIONS Taken together, these results demonstrated for the first time the in vivo chemopreventive efficacy and molecular mechanisms of dietary hydroxylated PMFs against AOM-induced colonic tumorigenesis.