Epidemiological and animal model studies suggest that a high intake of heme, present in red meat, is associated with an increased risk of colon cancer. The aim of this study was to elucidate the effects of dietary heme on colonic cell homeostasis in rats. Rats were fed a purified, humanized, control diet or a similar diet supplemented with 0.5 mmol heme/kg for 14 days. Fecal water cytolytic activity was determined with a bioassay, and colon epithelial cell proliferation was evaluated with (3)H-thymidine or 5-bromo-2'-deoxyuridine incorporation into DNA or by Ki-67 immunohistochemistry. Exfoliation of colonocytes was measured as the amount of rat DNA in feces, and caspase-3 expression and activity were measured to study colonic mucosal apoptosis. Dietary heme induced a >10-fold increased cytolytic activity of the fecal water and a 100-fold lower excretion of host DNA. Colons of heme-fed rats showed injured surface epithelium and an approximately 25% increase in crypt depth. Finally, dietary heme doubled colonocyte proliferation, shown by all three markers, but inhibited colonic mucosal apoptosis. In conclusion, our results demonstrate that dietary heme injures colonic surface epithelium, which is overcompensated by inhibition of apoptosis and hyperproliferation of cells in the crypts, resulting in crypt hyperplasia. This disturbed epithelial cell homeostasis might explain why a high intake of dietary heme is associated with an increased risk of colon cancer.