Cancer cells are usually under higher levels of oxidative stress compared to normal cells. We hypothesized that the introduction of additional reactive oxygen species (ROS) or the suppression of antioxidant activity may selectively enhance cancer cell killing by generating oxidative agents through stress overload or stress sensitization. The aim of this work was to test whether hydroxytyrosol (HT), one of the major polyphenolic constituents of extra virgin olive oil, could exert anti-cancer effects on human colon adenocarcinoma cells via its ability to induce apoptosis through ROS generation. HT exhibits preferential anti-proliferative effects on human colon cancer cells (DLD1 cells) but not on normal colon epithelial 1807 cells. HT causes oxidative stress, activates the phosphoinositide 3-kinase/Akt pathway, phosphorylates FOXO3a and then downregulates FOXO3a's target genes. Combined with SOD or catalase treatment, there are different responses in HT-treated DLD1 cells. The results support the hypothesis that the two main species of ROS, superoxide and H2O2, play different roles in cancer cell survival. The present work shows that HT induces apoptotic cell death and mitochondrial dysfunction by generating ROS in colon cancer cells. This research presents important evidence on the in vitro chemopreventive effect of HT and shows that the disruption of the intrinsic redox status promotes cancer cell death.