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ABSTRACT Glucose transporters are important for viability and infectivity of the disease-causing amastigote stages of Leishmania mexicana. The Δgt1-3 null mutant, in which the 3 clustered glucose transporter genes, GT1, GT2, and GT3, have been deleted, is strongly impaired in growth inside macrophages in vitro. We have now demonstrated that this null mutant is also impaired in virulence in the BALB/c murine model of infection and forms lesions considerably more slowly than wild-type parasites. Previously, we established that amplification of the PIFTC3 gene, which encodes an intraflagellar transport protein, both facilitated and accompanied the isolation of the original Δgt1-3 null mutant generated in extracellular insect-stage promastigotes. We have now isolated Δgt1-3 null mutants without coamplification of PIFTC3. These amplicon-negative null mutants are further impaired in growth as promastigotes, compared to the previously described null mutants containing the PIFTC3 amplification. In contrast, the GT3 glucose transporter plays an especially important role in promoting amastigote viability. A line that expresses only the single glucose transporter GT3 grows as well inside macrophages and induces lesions in animals as robustly as do wild-type amastigotes, but lines expressing only the GT1 or GT2 transporters replicate poorly in macrophages. Strikingly, GT3 is restricted largely to the endoplasmic reticulum in intracellular amastigotes. This observation raises the possibility that GT3 may play an important role as an intracellular glucose transporter in the infectious stage of the parasite life cycle. IMPORTANCE Glucose transport plays important roles for in vitro growth of insect-stage promastigotes and especially for viability of intramacrophage mammalian host-stage amastigotes of Leishmania mexicana. However, the roles of the three distinct glucose transporters, GT1, GT2, and GT3, in parasite viability inside macrophages and virulence in mice have not been fully explored. Parasite lines expressing GT1 or GT2 alone were strongly impaired in growth inside macrophages, but lines expressing GT3 alone infected macrophages and caused lesions in mice as robustly as wild-type parasites. Notably, GT3 localizes to the endoplasmic reticulum of intracellular amastigotes, suggesting a potential role for salvage of glucose from that organelle for viability of infectious amastigotes. This study establishes the unique role of GT3 for parasite survival inside host macrophages and for robust virulence in infected animals.