Malaria remains a devastating infectious disease, with symptoms caused by Plasmodium parasite invasion of erythrocytes. During blood-stage infection, parasites import and digest up to 80% of host cell hemoglobin. This massive catabolic process liberates vast amounts of heme, which parasites detoxify by sequestering it into crystalline hemozoin within an acidic digestive vacuole. Eukaryotic Plasmodium parasites also require heme as a metabolic cofactor within mitochondrial cytochromes. Despite access to abundant host heme, parasites retain a complete heme biosynthesis pathway, whose essentiality and properties have been the subject of considerable confusion and uncertainty. We have used mass spectrometry, photodynamic and chemical probes, and CRISPR/Cas9-based molecular genetics to dissect and understand parasite pathways for heme acquisition, evaluate heme biosynthesis as a potential antimalarial target, and directly test parasite reliance on respiratory cytochromes during blood-stage infection. These studies provide direct insight into parasite adaptations that equip them to survive and proliferate within human red blood cells and suggest multiple new therapeutic strategies.