Leishmania parasites alternate between flagellated promastigotes in sand flies and nonflagellated amastigotes in mammals, causing a spectrum of serious diseases. To survive, they must resist the harsh conditions in phagocytes (including acidic pH, elevated temperature, and increased oxidative/nitrosative stress) and evade the immune response. Recent studies have highlighted the importance of sphingolipid (SL) metabolism in Leishmania virulence. In particular, we have generated a Leishmania major iscl- mutant which is deficient in SL degradation but grows normally as promastigotes in culture. Importantly, iscl- mutants cannot induce pathology in either immunocompetent or immunodeficient mice yet are able to persist at low levels. In this study, we investigated how the degradation of SLs might contribute to Leishmania infection. First, unlike wild-type (WT) L. major, iscl- mutants do not trigger polarized T cell responses in mice. Second, like WT parasites, iscl- mutants possess the ability to downregulate macrophage activation by suppressing the production of interleukin-12 (IL-12) and nitric oxide. Third, during the stationary phase, iscl- promastigotes were extremely vulnerable to acidic pH but not to other adverse conditions, such as elevated temperature and oxidative/nitrosative stress. In addition, inhibition of phagosomal acidification significantly improved iscl- survival in murine macrophages. Together, these findings indicate that SL degradation by Leishmania is essential for its adaption to the acidic environment in phagolysosomes but is not required for the suppression of host cell activation. Finally, our studies with iscl- mutant-infected mice suggest that having viable, persistent parasites is not sufficient to provide immunity against virulent Leishmania challenge.