Toxoplasma gondii is an obligate intracellular protozoan that causes toxoplasmosis. Previous studies have shown that the perturbation of mitochondrial metabolism in T. gondii results in growth deficiency in host cells and lack of virulence in animals. Members of this Letm1 protein family are inner mitochondrial membrane proteins which play a role in potassium and hydrogen ion exchange. Letm1 has not been characterized in T. gondii. In this study, a potential TgLetm1 gene (TgGT1_288400) with Letm1-like protein domain coding sequence was identified in T. gondii. Indirect immunofluorescence assays suggested that TgLetm1 localized to the mitochondria in tachyzoites, as indicated by the colocalization with mitochondrial marker Mitotracker. TgLetm1 was found in the membrane fraction by western blot analysis. To investigate the role of TgLetm1 in T. gondii, we generated a tetracycline-inducible TgLetm1-knock-down mutant. The conditional deletion of TgLetm1 resulted in mitochondrial swelling. Functional studies showed that the conditional deletion of TgLetm1 resulted in growth inhibition, deficiency in invasion and replication, and lack of virulence in mice.