The consumption of red meat is probably carcinogenic to humans and is associated with an increased risk to develop colorectal cancer (CRC). Red meat contains high amounts of heme iron, which is thought to play a causal role in tumor formation. In this study, we investigated the genotoxic and cytotoxic effects of heme iron (i.e., hemin) versus inorganic iron in human colonic epithelial cells (HCEC), human CRC cell lines and murine intestinal organoids. Hemin catalyzed the formation of reactive oxygen species (ROS) and induced oxidative DNA damage as well as DNA strand breaks in both HCEC and CRC cells. In contrast, inorganic iron hardly affected ROS levels and only slightly increased DNA damage. Hemin, but not inorganic iron, caused cell death and reduced cell viability. This occurred preferentially in non-malignant HCEC, which was corroborated in intestinal organoids. Both hemin and inorganic iron were taken up into HCEC and CRC cells, however with differential kinetics and efficiency. Hemin caused stabilization and nuclear translocation of Nrf2, which induced heme oxygenase-1 (HO-1) and ferritin heavy chain (FtH). This was not observed after inorganic iron treatment. Chemical inhibition or genetic knockdown of HO-1 potentiated hemin-triggered ROS generation and oxidative DNA damage preferentially in HCEC. Furthermore, HO-1 abrogation strongly augmented the cytotoxic effects of hemin in HCEC, revealing its pivotal function in colonocytes and highlighting the toxicity of free intracellular heme iron. Taken together, this study demonstrated that hemin, but not inorganic iron, induces ROS and DNA damage, resulting in a preferential cytotoxicity in non-malignant intestinal epithelial cells. Importantly, HO-1 conferred protection against the detrimental effects of hemin.