Lathosterol oxidase (LSO) catalyzes the formation of the C-5–C-6 doublebond in the synthesis of various types of sterols in mammals, fungi, plants, and pro-tozoa. InLeishmaniaparasites, mutations inLSOor other sterol biosynthetic genesare associated with amphotericin B resistance. To investigate the biological roles ofsterol C-5–C-6 desaturation, we generated anLSO-null mutant line (lso)inLeishma-nia major, the causative agent for cutaneous leishmaniasis.lsoparasites lacked theergostane-based sterols commonly found in wild-typeL. majorand instead accumu-lated equivalent sterol species without the C-5–C-6 double bond. These mutant par-asites were replicative in culture and displayed heightened resistance to amphoteri-cin B. However, they survived poorly after reaching the maximal density and werehighly vulnerable to the membrane-disrupting detergent Triton X-100. In addition,lsomutants showed defects in regulating intracellular pH and were hypersensitiveto acidic conditions. They also had potential alterations in the carbohydrate compo-sition of lipophosphoglycan, a membrane-bound virulence factor inLeishmania. Allthese defects inlsowere corrected upon the restoration of LSO expression. To-gether, these findings suggest that the C-5–C-6 double bond is vital for the struc-ture of the sterol core, and while the loss of LSO can lead to amphotericin B resis-tance, it also makesLeishmaniaparasites vulnerable to biologically relevant stress.