Although iodization of salt is the most common method used to obtain iodine-enriched food, iodine deficiency disorders are still a global health problem and profoundly affect the quality of human life. Iodine is required for the synthesis of thyroid hormones, which are crucial regulators of human metabolism, cell growth, proliferation, apoptosis and have been reported to be involved in carcinogenesis. In this study, for the first time, we evaluated the effect of iodine-biofortified lettuce on transcriptomic profile of Caco-2 cancer cell line by applying the Whole Human Genome Microarray assay. We showed 1326 differentially expressed Caco-2 transcripts after treatment with iodine-biofortified (BFL) and non-fortified (NFL) lettuce extracts. We analysed pathways, molecular functions, biological processes and protein classes based on comparison between BFL and NFL specific genes. Iodine, which was expected to act as a free ion (KI-NFL) or at least in part to be incorporated into lettuce macromolecules (BFL), differently regulated pathways of numerous transcription factors leading to different cellular effects. In this study we showed the inhibition of Caco-2 cells proliferation after treatment with BFL, but not potassium iodide (KI), and BFL-mediated induction of mitochondrial apoptosis and/or cell differentiation. Our results showed that iodine-biofortified plants can be effectively used by cells as an alternative source of this trace element. Moreover, the observed differences in action of both iodine sources may suggest a potential of BFL in cancer treatment.