Mitochondrial iron is indispensable for heme biosynthesis and iron–sulfur cluster assembly. Several mitochondrial transmembrane proteins have been implicated to function in the biosynthesis of heme and iron–sulfur clusters by transporting reaction intermediates. However, several mitochondrial proteins related to iron metabolism remain uncharacterized. Here, we show that human sideroflexin 2 (SFXN2), a member of the SFXN protein family, is involved in mitochondrial iron metabolism. SFXN2 is an evolutionarily conserved protein that localized to mitochondria via its transmembrane domain. SFXN2-knockout (KO) cells had an increased mitochondrial iron content, which was associated with decreases in the heme content and heme-dependent enzyme activities. By contrast, the activities of iron–sulfur cluster-dependent enzymes were unchanged in SFXN2-KO cells. Moreover, abnormal iron metabolism impaired mitochondrial respiration in SFXN2-KO cells and accelerated iron-mediated death of these cells. Our findings demonstrate that SFXN2 functions in mitochondrial iron metabolism by regulating heme biosynthesis.