Heme is an essential cofactor for several biological processes in most organisms. It is synthesized in a highly conserved enzymatic pathway. Despite its role in maintaining cellular homeostasis, the molecular mechanisms underlying heme regulation are poorly understood. To address this knowledge gap, we investigated the interactome and mechanism of ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. First, using affinity purification-mass spectrometry and bioinformatics tools, we mapped the FECH interactome in human and murine cell lines to reveal a comprehensive network of known and novel FECH protein partners, some of which are involved in the heme biosynthetic process and mitochondrial organization. Our data highlighted the association of the FECH interactome with biological processes including the tricarboxylic acid cycle, which provides succinyl-CoA, a precursor for heme synthesis. We also identified proteins that form the core components of the FECH interactome, including progesterone receptor component 1, a heme-binding protein previously proposed as a heme chaperone. Second, to elucidate the mechanism of FECH, we carried out comparative studies using the protozoan Leishmania FECH (LFech) and FECH from other prokaryotic and eukaryotic organisms. Using molecular dynamic simulations, we discovered that different residues appear to be involved in heme and protoporphyrin IX binding in LFech compared to human FECH (HsFECH). The project also unveiled a previously unappreciated role of a conserved phenylalanine residue in LFech, which may play a crucial role in the metalation of protoporphyrin IX, like the methionine residue in HsFECH. Put together, our studies uncovered factors that regulate heme biosynthesis and the unique structural features of LFech. These findings have significant implications for future studies on heme-associated diseases and the development of targeted therapies.