Heme is an essential co-factor for many critical cellular processes. The protozoan parasite Leishmania amazonensis is a heme auxotroph and must acquire heme from the environment. One pathway of heme acquisition is through Leishmania Heme Response 1 (LHR1), a heme transporter localized to the plasma membrane and acidic intracellular compartments. In this work we further characterize LHR1 and the mechanism by which it promotes heme uptake. We show that overexpression of LHR1 in Leishmania amazonensis increases the total parasite intracellular heme pool, and that expression in Saccharomyces cerevisiae promotes uptake of the heme analog Zinc Mesoporphyrin IX (ZnMP). Our results indicate that heme binding to LHR1 is pH independent, whereas heme transport by the parasites is more efficient under acidic conditions. To examine the molecular mechanisms responsible for LHR1 heme transport, we performed a mutagenesis analysis of LHR1. We show that three key tyrosines residues, Tyr-18, Tyr-80, and Tyr-129, located in predicted transmembrane domains near the cytoplasmic leaflet of the plasma membrane, are important for heme transport. Although the mutant proteins appear to not affect promastigote growth, they have a profound inhibitory effect on intracellular amastigote replication in macrophages, and are necessary for virulence in vivo. Finally, we also examine the differential regulation of LHR1 expression in a visceralizing species, Leishmania chagasi, compared to L. amazonensis, a species that causes cutaneous lesions. L. chagasi has higher amounts of LHR1 transcripts than Leishmania amazonensis under heme-depleted conditions, and uptakes ZnMP faster and to a greater extent than Leishmania amazonensis. This differential regulation of LHR1 may be due to differences in the gene 3’ Untranslated Regions (UTRs) between the two species. This works adds to our understanding of the critical process of heme transport and its role in Leishmania virulence.