Heme is an essential cofactor involved in various biological processes including oxygen transport, xenobiotic detoxification, oxidative metabolism, gas sensing, circadian rhythm, signal transduction, microRNA processing and thyroid hormone synthesis. Heme is also an essential nutrient for parasites and is the major dietary iron source for humans. Despite our extensive understanding of the mechanisms of heme synthesis and degradation in eukaryotes, little is known as to how heme is transported and trafficked in eukaryotes. Recently, CeHRG-1 and CeHRG-4 were identified as the first bona fide heme importers/transporters using the heme auxotroph, Caenorhabditis elegans. To gain mechanistic insights into the heme transport function of HRG-1-related proteins, we conducted a structure-function analysis of CeHRG-1 and CeHRG-4 by exploiting yeast mutants that are genetically defective in heme synthesis. Our studies reveal that HRG-1-related proteins transport heme across membranes through the coordinated actions of strategically placed amino acids that are topologically conserved in both, the worm and human proteins. To further dissect the functional elements that dictate their intracellular localization, we generated a series of chimeras by swapping the amino and carboxy terminal segments of CeHRG-1 and CeHRG-4. Our analysis in yeast and mammalian cells demonstrate that the C-terminal domains are essential for membrane localization of the protein, while the N-terminal domains are important for proper function, and plausibly multimerization of HRG-1-related proteins. Currently, there are no pharmacological means to aid in the study of the cellular and physiological roles of eukaryotic heme transporters. We, for the first time, developed and executed a high-throughput screen of 233,360 compounds, to identify potential antagonists of HRG-1-related proteins by utilizing parasite heme transporters as the primary screening bait. Subsequent study in parasites will provide novel drug candidates against helminths that infect humans, livestock, and plants, as well as against genetic disorders of heme and iron metabolism in humans. Taken together, results from our studies will significantly advance novel functional and therapeutic insights into HRG-1 mediated heme transport in health and disease.