Lipolysis of complex natural lipoproteic assemblies such as milk fat globules is particularly frequent during neonatal digestion1. Such lipolysis proceeds at acid pH and requires a rapid adsorption of the enzyme onto the external membrane enveloping the substrate before the onset of catalytic activity2. The interactions governing this adsorption are not fully elucidated. Our objective was thus to unravel the involved interactions and precise gastric lipase lateral distribution in model membranes of milk fat globules presenting liquid phase coexistence. Recombinant dog gastric lipase (rDGL) was used as model of the human gastric lipase. Ellipsometry, tensiometry and atomic force microscopy were used to get an insight on the ability of rDGL to get inserted into the lipid membrane. Lipid Langmuir films were used to mimic the outer leaflet of the external membrane of the milk fat globule. Different lipid mixtures and natural extracts of cow’s buttermilk with variable physical phases, surface charge and lateral packing were tailored to identify the nature of the enzyme/membrane interactions. We showed that: 1.rDGL is characterized by a high affinity for the lipid/liquid interface as revealed by adsorption equilibrium coefficients. Nevertheless, a large amount of molecules is located close to the interface but a limited number is inserted; 2. rDGL partitions toward liquid expansed phase and at phase boundaries, gets adsorbed at three levels of insertion (h1~5.5 nm, h2<4 nm, and h3<2 nm) and strongly impacts on lipid phase lateral organization (Figure); 3. Addition of local negative charges using phosphatidylserine reinforced adsorption; hence besides hydrophobic interactions, rDGL adsorption is favored by electrostatic interactions which were investigated through surface potential modelling. The massive amount of gastric lipase located close to the substrate may favor rapid prelipolysis before gastric emptying, the release of short to medium chain fatty acids as a rapid source of energy and the subsequent action of pancreatic lipase. Investigation of the mechanism of adsorption of gastric lipase in supported multilayers will be the next step of our research to further approach the complexity of the milk fat globule.