Extracellular vesicles (EVs) (exossomes, microvesicles and apoptotic bodies) have been well acknowledged as mediators of intercellular communications in prokaryotes and eukaryotes. Lipids are essential molecular components of EVs but at the moment the knowledge about the lipid composition and the function of lipids in EVs is limited and as for now none lipidomic studies in Giardia EVs was described. Therefore, the focus of the current study was to conduct, for the first time, the characterization of the polar lipidome, namely phospholipid and sphingolipid profiles of G. lamblia trophozoites, microvesicles (MVs) and exosomes, using C18-Liquid Chromatography-Mass Spectrometry (C18-LC-MS) and Tandem Mass Spectrometry (MS/MS). A total of 162 lipid species were identified and semi-quantified, in the trophozoites, or in the MVs and exosomes belonging to 8 lipid classes, including the phospholipid classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), cardiolipins (CL), the sphingolipid classes sphingomyelin (SM) and ceramides (Cer), and cholesterol (ST), and 3 lipid subclasses that include lyso PC (LPC), lyso PE (LPE) and lyso PG (LPG), but showing different abundances. This work also identified, for the first time, in G. lamblia trophozoites, the lipid classes CL, Cer and ST and subclasses of LPC, LPE and LPG. Univariate and multivariate analysis showed clear discrimination of lipid profiles between trophozoite, exosomes and MVs. The principal component analysis (PCA) plot of the lipidomics dataset showed clear discrimination between the three groups. Future studies focused on the composition and functional properties of Giardia EVs may prove crucial to understand the role of lipids in host-parasite communication, and to identify new targets that could be exploited to develop novel classes of drugs to treat giardiasis.