Human hemoglobin is an essential protein， whose main function as an oxygen carrier is indispensable for life. Hemoglobin is a cofactor-containing protein with heme as prosthetic group. Same as in humans， heme is synthesized in many organisms in a complex pathway involving two cellular compartments (mitochondria and cytosol)， which is tightly regulated. Red blood cells (erythrocytes) are specialized and adapted for production and transport of the hemoglobin molecules. In addition to oxygen binding， hemoglobin can participate in a variety of chemical reactions by its iron and heme and may become toxic when released from erythrocytes. Hemoglobin is a major target for the development of blood substitutes/oxygen carriers， and therefore its microbial production is attractive， as it may provide a cheap and reliable source of human hemoglobin. Significant efforts have been dedicated to this task for the last three decades. Moreover since the first generation of cell-free blood substitutes based on unmodified hemoglobin failed human trials， mutant forms became of great interest.In this chapter we summarize the existing knowledge about human hemoglobin， challenges of its microbial production， and its improvement， with a particular focus upon yeast as production host.