Red meat consumption is associated with an increased colon cancer risk. Heme, present in red meat, injures the colon surface epithelium by luminal cytotoxicity and reactive oxygen species. This surface injury is compensated by hyperproliferation and hyperplasia of crypt cells, which was induced by a changed surface to crypt signalling as recently described. It is unknown whether the change in signaling is caused by cytotoxic stress and/or by oxidative stress, as these processes were never studied separately. Therefore, the aim of this study was to determine the possible differential effects of dietary heme on these luminal stressors and their impact on the colonic mucosa after 2, 4, 7 and 14 days of heme feeding. Mice received a purified humanized control diet or this diet supplemented with 0.2 µmol heme/g. Oxidative stress was measured as Thiobarbituric Acid Reactive Substances (TBARS) in fecal water. Cytotoxicity of fecal water was quantified with a bioassay. Epithelial cell proliferation was determined by Ki67 immunohistochemistry and mucosal responses were further studied in detail by whole genome transcriptomics. Dietary heme caused instantaneous and delayed changes in the luminal contents which were reflected in the mucosa. Instantaneous, there was an increase in reactive oxygen species leading to increased levels of lipid peroxidation products. Mucosal gene expression showed an instantaneous antioxidant response and PPAR target gene activation. After day 4 cytotoxicity of the colonic contents was increased and hyperproliferation was initiated, indicating that cytotoxicity was causal for the initiation of hyperproliferation. Several oncogenes were activated and tumor protein 53 was inhibited. In conclusion, dietary heme caused an instantaneous production of reactive oxygen species in mouse colon. A lag time was observed in the formation of cytotoxicity which coincided with the initiation hyperproliferation.