Previously, our research provided evidence that exposure of gastric and colon cancer cells to extracts from iodine-biofortified lettuce leads to a reduction of cell viability and proliferation through cell cycle arrest and upregulation of pro-apoptotic genes. The aim of the present study was to determine the potential cellular mechanisms of induction of cell death in human gastrointestinal cancer cell lines after treatment with iodine-biofortified lettuce. We demonstrated that extracts from lettuce enriched with iodine induce apoptosis in gastric AGS and colon HT-29 cancer cells and the mechanism of programmed cell death may be triggered and executed through different signaling pathways, depending on the type of cells. Western blot analysis revealed that iodine-fortified lettuce leads to cell death through the release of cytochrome c to the cytosolic fraction and activation of the primary drivers of apoptosis: caspase-3, caspase-7, and caspase-9. Furthermore, we have reported that apoptotic effects of lettuce extracts may be mediated by poly (ADP-ribose) polymerase (PARP) and activation of pro-apoptotic Bcl-2 family proteins such as Bad, Bax, and BID. We also observed mitochondrial dysfunction with the dissipation of the mitochondrial membrane potential in cells exposed to lettuce extracts. Taken together, these results indicate that the organic form of iodine such as 5-ISA and 3,5-diISA is an important factor in the activation of intrinsic mitochondrial apoptotic pathway in AGS and HT-29 cancer cells in a p53-independent manner.