Heme is an essential cofactor, signaling molecule, and nutritional source of iron. Despite its tremendous importance in biology, the mechanisms that regulate heme transport and trafficking for use as a cofactor or signaling molecule are poorly understood. In order to elucidate cellular heme trafficking and signaling pathways, we have developed genetically encoded ratiometric fluorescent sensors to image heme biodistribution and dynamics and hemoproteomics approaches to identify new heme binding proteins. We have applied these tools to identify and characterize: novel targets of heme signaling, heme homeostatic factors, and mechanisms of heme acquisition by microbial pathogens. In this presentation, I will highlight short vignettes describing our efforts to illuminate heme homeostatic and signaling pathways in both eukaryotes and prokaryotes.