ATP-binding cassette (ABC) transporters represent an important family of membrane proteins involved in drug resistance and other biological activities. The present work reports the characterization of the first ABC subfamily G (ABCG)-like transporter, LiABCG4, in the protozoan parasite Leishmania. LiABCG4 localized mainly to the parasite plasma membrane. Overexpression of this half-transporter reduced the accumulation of phosphatidylcholine analogues and conferred resistance to alkyl-phospholipids. Likewise, when expressed in Saccharomyces cerevisiae, the protein localized to the yeast plasma membrane and conferred resistance to alkyl-phospholipids. Post-Golgi secretory vesicles isolated from a LiABCG4-overexpressing yeast mutant contained the leishmanial ABC transporter and exhibited ATP-dependent, vanadate-sensitive transport of phosphatidylcholine analogues from the cytosolic to the lumenal leaflet of the vesicle membrane. Cross-linking showed dimerization of LiABCG4. These results suggest that LiABCG4 is involved in the active transport of phosphatidylcholine and resistance to alkyl-phospholipids in Leishmania.