Abstract We investigated the anti-proliferative effects of an olive oil polyphenolic extract on human colon adenocarcinoma cells. Analysis indicated that the extract contained hydroxytyrosol, tyrosol and the various secoiridoid derivatives, including oleuropein. This extract exerted a strong inhibitory effect on cancer cell proliferation, which was linked to the induction of a G2/M phase cell cycle block. Following treatment with the extract (50 microg/ml) the number of cells in the G2/M phase increased to 51.82+/-2.69% relative to control cells (15.1+/-2.5%). This G2/M block was mediated by the ability of olive oil polyphenols (50 microg/ml) to exert rapid inhibition of p38 (38.7+/-4.7%) and CREB (28.6+/-5.5%) phosphorylation which led to a downstream reduction in COX-2 expression (56.9+/-9.3%). Our data suggest that olive oil polyphenols may exert chemopreventative effects in the large intestine by interacting with signalling pathways responsible for colorectal cancer development.