Human neutrophilpeptide 1 (HNP1) is a human defensin with antimicrobial activity against different bacteria (both Gram-positive and negative), fungi, and viruses. HNP1 is stored in the cytoplasmic azurophilic granules of neutrophils. To elucidate the mode of action of this antimicrobial peptide, studies based on its lipid selectivity were carried out. Large unilamellar vesicles with different lipid compositions were used as biomembranes model systems (mammal, fungal, and bacterial models). Changes on the intrinsic fluorescence of HNP1 upon membrane binding/insertion show that HNP1 has quite distinct preferences for mammalian and fungal membrane model systems. HNP1 showed low interaction with glucosylceramide rich membranes, but high sterol selectivity: it has a higher partition for ergosterol-containing membranes (as fungal membranes) and lower interaction with cholesterol-containing membranes (as in mammalian cells). These results reveal that lipid selectivity is a determinant step for HNP1 action. Fluorescence quenching data obtained using acrylamide indicate that HNP1 interacts with membranes without a full insertion in the lipid bilayer. Generalized polarization of laurdan indicates a change in membrane fluidity in the presence of HNP1 for POPC membranes but not for ergosterol-enriched membranes.