Heme is a prosthetic group in proteins that perform diverse biological functions including respiration, gas sensing, xenobiotic detoxification, cell differentiation, circadian clock control and micro RNA processing. In most eukaryotes, heme is synthesized through a multi-step pathway with defined intermediates that are highly conserved through evolution. Despite our extensive knowledge about heme biosynthesis and degradation, the molecules and pathways involved in intracellular heme trafficking are unknown, primarily due to the inability to dissociate the tightly regulated processes of heme biosynthesis and degradation from intracellular trafficking events. Caenorhabditis elegans and related helminths are natural heme auxotrophs that rely solely on exogenous heme for normal development and reproduction. We performed a genome-wide microarray analysis and identified 288 genes that are regulated by heme at the transcriptional level in C. elegans. Here, we characterize two heme-responsive genes, hrg-1 and its paralog hrg-4, that are highly upregulated at low heme concentrations and demonstrate that HRG-1 and HRG-4 are heme transporters. Depletion of hrg-1 and hrg-4 in worms by RNAi results in the disruption of organismal heme homeostasis and abnormal response to heme analogs. HRG-4 traffics to the plasma membrane, and HRG-1 localizes to endo-lysosomal compartments. While hrg-4 appears to be specific to worms, hrg-1 has homologs in vertebrates. Knock-down of hrg-1 in zebrafish results in severe anemia and profound developmental defects, which are fully rescued by worm hrg-1. Human and worm HRG-1 proteins localize together. CeHRG-1, hHRG1 and CeHRG-4 all bind and transport heme. To further understand the in vivo functions of hrg-1 and hrg-4, we characterize the genetic deletions of these genes in C. elegans. Preliminary experiments suggest that the deletion mutants respond abnormally to heme analogs, although these results do not phenocopy the RNAi knock-down studies. We speculate that the deletion strains may have developed compensatory mechanisms in response to the genetic lesions in hrg-1 and hrg-4. Taken together, the studies described herein lay the foundation for identifying the molecular mechanisms for heme transport by the HRG-1 proteins in metazoans and delineating the heme trafficking pathways in C. elegans.