Considering the growing number of cancer cases around the world, natural products from the diet that exhibit potential antitumor properties are of interest. Our previous research demonstrated that fortification with iodine compounds is an effective way to improve the antioxidant potential of lettuce. The purpose of the present study was to evaluate the effect of iodine-biofortified lettuce on antitumor properties in human gastrointestinal cancer cell lines, gastric AGS and colon HT-29. Our results showed that extracts from iodine-biofortified lettuce reduce the viability and proliferation of gastric and colon cancer cells. The extracts mediated cell cycle arrest which was accompanied by inactivation of anti-apoptotic Bcl-2 and activation of caspases, as assessed by flow cytometry. However, extracts from lettuce fortified with organic forms of iodine acted more effectively than extracts from control and KIO-enriched plants. Using quantitative PCR, we detected the increase in pro-apoptotic genes and in AGS cells whereas up-regulation of cell cycle progression inhibitor and downregulation of pro-proliferative in HT-29 cells. Interestingly, lettuce extracts led to down-regulation of pro-survival and protooncogenic , which was consistent for extracts of lettuce fortified with organic form of iodine, 5-ISA, in both cell lines. downregulation in HT-29 colon cancer cells was associated with upregulation upon 5-ISA-fortified lettuce extracts, which provides a link to the epigenetic regulation of tumor suppressor genes by RB/MDM2 pathway. Indeed, tumor suppressor gene was hypomethylated and upregulated in HT-29 cells treated with 5-ISA-fortified lettuce. Control lettuce exerted similar effects on RB/MDM2 pathway and epigenetic activation in HT-29 cells. Our findings suggest that lettuce as well as lettuce fortified with organic form of iodine, 5-ISA, may exert epigenetic anti-cancer effects that can be cancer type-specific.