The accessibility of sterols in mammalian cells to exogenous sterol-binding agents has been well-described previously, but sterol accessibility in distantly related protozoa is unclear. The human pathogenLeishmania majoruses sterols and sphingolipids distinct from those used in mammals. Sterols in mammalian cells can be sheltered from sterol-binding agents by membrane components, including sphingolipids, but the surface exposure of ergosterol inLeishmaniaremains unknown. Here, we used flow cytometry to test the ability of theL. majorsphingolipids inositol phosphorylceramide (IPC) and ceramide to shelter ergosterol by preventing binding of the sterol-specific toxins streptolysin O and perfringolysin O and subsequent cytotoxicity. In contrast to mammalian systems, we found thatLeishmaniasphingolipids did not preclude toxin binding to sterols in the membrane. However, we show that IPC reduced cytotoxicity and that ceramide reduced perfringolysin O- but not streptolysin O-mediated cytotoxicity in cells. Furthermore, we demonstrate ceramide sensing was controlled by the toxin L3 loop, and that ceramide was sufficient to protectL. majorpromastigotes from the anti-leishmaniasis drug amphotericin B. Based on these results, we propose a mechanism whereby pore-forming toxins engage additional lipids like ceramide to determine the optimal environment to sustain pore formation. Thus,L.majorcould serve as a genetically tractable protozoan model organism for understanding toxin-membrane interactions.