The traditional Mediterranean diet is thought to represent a healthy lifestyle; especially given the incidence of several cancers including colorectal cancer is lower in Mediterranean countries compared to Northern Europe. Olive oil, a central component of the Mediterranean diet, is believed to beneficially affect numerous biological processes. We used phenols extracted from virgin olive oil on a series of in vitro systems that model important stages of colon carcinogenesis. The effect the extract on DNA damage induced by hydrogen peroxide was measured in HT29 cells using single cell microgel-electrophoresis. A significant anti-genotoxic linear trend (p = 0.011) was observed when HT29 cells were pre-incubated with olive oil phenols (0, 5, 10, 25, 50, 75, 100 μg/ml) for 24 hr, then challenged with hydrogen peroxide. The olive oil phenols (50, 100 μg/ml) significantly (p = 0.004, p = 0.002) improved barrier function of CACO2 cells after 48 hr as measured by trans-epithelial resistance. Significant inhibition of HT115 invasion (p < 0.01) was observed at olive oil phenols concentrations of 25, 50, 75, 100 μg/ml using the matrigel invasion assay. No effect was observed on HT115 viability over the concentration range 0, 25, 50 75, 100 μg/ml after 24 hr, although 75 and 100 μg/ml olive oil phenols significantly inhibited HT115 cell attachment (p = 0.011, p = 0.006). Olive oil phenols had no significant effect on metastasis-related gene expression in HT115 cells. We have demonstrated that phenols extracted from virgin olive oil are capable of inhibiting several stages in colon carcinogenesis in vitro. © 2005 Wiley-Liss, Inc.