Psd2 is a 47 aminoacids pea defensin, which inhibits the growth of fungal species by an uncharacterized mechanism of action. In this work, the interaction of Psd2 with model membranes mimicking the lipid composition of different organisms was evaluated. Protein-lipid overlay assays indicated that Psd2 bound to Fusarium solani glucosylceramide (GlcCerF. solani) and ergosterol (Erg), besides others PIPs, suggesting that these lipids may play an important role in the Psd2 antifungal activity. No reaction with cholesterol, a typical mammalian membrane lipid, was observed. In agreement with these observations, Surface Plasmon Resonance analysis showed that Psd2 interacts with higher affinity to vesicles (SUVs) containing GlcCer and ergosterol than those composed of cholesterol. Fluorescence spectroscopy lipid partition experiments confirmed the Psd2 preference for large unilamellar vesicles (LUVs) containing POPC:GlcCerF. solani 70:30 and also for POPC:GlcCerF.solani:Erg 50:20:30 that mimic fungal rafts environment The acquired partition coefficients (Kp) were 52 ± 5 and 103 ± 20 x 103 respectively, higher then that when cholesterol-containing membranes were used (Kp = 13 ± 1 x 103). Furthermore, we showed that not only the GlcCer presence, but also some of this particular structural elements, as the presence of double bonds between C8-C9 and a methyl group at position C9 of the sphingoid base backbone, were relevant for Psd2 antifungal activity. These in vitro results were in harmony with the high inhibitory effect of Psd2 in fungal growth, especially in species that synthesize GlcCer, and its lack of death activity on mammalian blood cells.