It has recently been shown that the iron isotopic composition of blood differs between individuals and sexes, which is supposed to reflect individual differences in iron metabolism. We hypothesized that patients suffering from hereditary hemochromatosis would demonstrate alterations in the iron isotopic composition of blood due to persistent up-regulation of intestinal iron absorption. Blood from 30 patients with homozygous C282Y hemochromatosis was analyzed for iron isotopic composition by a newly developed technique using multicollector inductively coupled plasma mass spectrometry (MC-ICP-MS). Blood of patients with hemochromatosis is characterized by a higher 56Fe/54Fe isotope ratio than blood of healthy individuals, which are either members of an age-matched control group (n = 10; P < .001) or young adults (n = 36; P < .001). In patients with hereditary hemochromatosis, the 56Fe/54Fe isotope ratio of blood significantly correlates with total-body iron accumulation, severity of clinical disease, and the need for regular phlebotomies to prevent iron reaccumulation. We conclude that blood of patients with hereditary hemochromatosis contains more of the heavier iron isotopes than blood of healthy individuals. The primary determinant of the iron isotopic composition of blood appears to be isotope-sensitive iron absorption in the intestine and the efficiency of this process.