The synthesis of the major phospholipids, including those that play an essential role in Leishmania virulence, initiates with the acylation of glycerol-3-phosphate and dihydroxyacetonephosphate at the sn-1 position by glycerol-3-phosphate and dihydroxyacetonephosphate acyltransferases respectively. In this study, we show that Leishmania major promastigotes express a single glycerol-3-phosphate acyltransferase activity important for triacylglycerol synthesis but not essential for virulence. The encoding gene, LmGAT, expressed in yeast results in full complementation of the lethality of a mutant, gat1Deltagat2Delta, lacking glycerol-3-phosphate activity. Biochemical analyses revealed that LmGAT is a low-affinity glycerol-3-phosphate acyltransferase and exhibits higher specific activity with unsaturated long fatty acyl-CoA donors. A L. major null mutant, Deltalmgat/Deltalmgat, was created and a thorough analysis of its lipid composition was performed. Deletion of LmGAT resulted in a complete loss of Leishmania glycerol-3-phosphate acyltransferase activity and a major reduction in triacylglycerol synthesis. Consistent with the specificity of LmGAT for glycerol-3-phosphate but not dihydroxyacetonephosphate, Deltalmgat/Deltalmgat mutant expressed normal levels of the ether-lipid derivatives and virulence factors, lipophosphoglycan and GPI-anchored proteins, gp63, and its virulence was not affected in mice.