Heme is an essential cofactor for diverse biological processes such as oxygen transport, xenobiotic detoxification, and circadian clock control. Since free heme is hydrophobic and cytotoxic, we hypothesize that within eukaryotic cells, specific trafficking pathways exist for the delivery of heme to different subcellular destinations where hemoproteins reside. To identify molecules that may be involved in heme homeostasis, we conducted a C. elegans microarray experiment on RNA extracted from worms grown at different concentrations of heme in axenic liquid medium. Analysis of the microarrays revealed that the mRNA levels of heme-responsive gene-2 (hrg-2) and hrg-3 increased more than 70 fold when worms were grown at 4 µM compared to 20 µM heme. hrg-2 is expressed in hypodermal tissues in the worm, and the protein localizes to the endoplasmic reticulum and the apical plasma membrane. In vitro hemin agarose pull-down experiments indicate that HRG-2 binds heme. Deletion of hrg-2 in C. elegans leads to reduced growth rate at low heme. Moreover, expression of HRG-2 in hem1δ, a heme-deficient yeast strain, results in growth rescue at submicromolar concentrations of exogenous heme. These results indicate that HRG-2 may either directly participate in heme uptake or facilitate heme delivery to another protein. Unlike hrg-2, hrg-3 is exclusively expressed in the worm intestine under heme deficiency. Following its synthesis, HRG-3 is secreted into the body cavity pseudocoelom. Deletion of hrg-3 results in increased heme levels in the worm intestine, suggesting that HRG-3 may function in intercellular heme transport in C. elegans. To identify the functional network or pathways for HRG-2 and HRG-3, we performed a genome-wide microarray analysis using RNA samples prepared from the worms grown at different concentrations of heme and oxygen. The results showed that a total of 446 genes were transcriptionally altered by heme and/or oxygen. Among them, 41 and 29 genes exhibited similar expression profiles to hrg-2 and hrg-3, respectively. We postulate that these genes may function in conjunction with hrg-2 and hrg-3. Taken together, we have identified two novel heme-responsive genes in metazoa that may play critical roles in modulating organismal heme homeostasis in C. elegans.