Proteins are considered to be amphitropic when translocation to/from biomembranes: i ) is required for function; ii ) occurs transiently and reversibly from the cytoplasm; and iii ) involves interaction with the lipid bilayer ( 1-3 ). Glycolipid transfer proteins (GLTPs) comprise a structurallyunique amphitropic protein superfamily defi ned by their ability to reversibly interact with membranes to achieve selective transport of glycosphingolipids ( 4 ). Human GLTP resides in the cytosol ( 5 ), but is capable of delivering glucosylceramide to the cytosolic face of the plasma membrane ( 6, 7 ) and contains a nonclassical diphenylalanine-in-anacidic-tract targeting sequence to the endoplasmic reticulum ( 8 ). GLTPs occur widely among eukaryotes ( 4, 9 ) and GLTP-like domains serve as key functional regions in larger human proteins, i.e., FAPP2 ( 10, 11 ). The GLTPfold is dominated by ␣ -helices, lacks intramolecular disulfi des, and uses a two-layer "sandwich" motif to form a single glycolipid binding site consisting of a surface-localized sugar headgroup recognition center and an expandable hydrophobic pocket for accommodation of the ceramide hydrocarbon chains of the glycolipid ( 12-17 ). During glycolipid transfer, GLTP interacts with membranes transiently and in a minimally perturbing manner ( 18, 19 ) via a membrane interaction domain formed by a cluster of Trp, Tyr, Lys, and nonpolar residues on the GLTP surface Abstract Among amphitropic proteins, human glycolipid transfer protein (GLTP) forms a structurally-unique fold that translocates on/off membranes to specifi cally transfer glycolipids. Phosphatidylcholine (PC) bilayers with curvature-induced packing stress stimulate much faster glycolipid intervesicular transfer than nonstressed PC bilayers raising questions about planar cytosol-facing biomembranes being viable sites for GLTP interaction. Herein, GLTP-mediated desorption kinetics of fl uorescent glycolipid (tetramethyl-boron dipyrromethene (BODIPY)-label) from lipid monolayers are assessed using a novel microfl uidics-based surface balance that monitors lipid lateral packing while simultaneously acquiring surface fl uorescence data. At biomembrane-like packing (30-35 mN/m), GLTP uptake of BODIPY-glycolipid from POPC monolayers was nearly nonexistent but could be induced by reducing surface pressure to mirror packing in cur vature-stressed bilayers. In contrast, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) matrices supported robust BODIPY-glycolipid uptake by GLTP at both high and low surface pressures. Unexpectedly, negativelycharged cytosol-facing lipids, i.e., phosphatidic acid and phosphatidylserine, also supported BODIPY-glycolipid uptake by GLTP at high surface pressure. Remarkably, including both 1-palmitoyl-2-oleoyl-sn -glycero-3-phosphate (5 mol%) and POPE (15 mol%) in POPC synergistically activated GLTP at high surface pressure. Our study shows that matrix lipid headgroup composition, rather than molecular packing per se, is a key regulator of GLTP-fold function while demonstrating th...