Lipids provide most calories necessary for newborn growth and development. Lipid digestion is initiated by the Human Gastric lipase (HGL) which is already mature at birth and is a key lipase in neonatal digestion. HGL is supposed to attack directly Native Milk Fat Globules (NMFG) found in unprocessed milk despite their complex supramolecular structure and protective trilayered membrane. However, the specific action of HGL on this natural substrate has not been studied in detail, nor directly compared with the same substrate after processing into infant formula. This study aimed at comparing the digestion kinetics of NMFG with that of processed lipid droplets of a model infant formula, during gastric in vitro digestion and at immunolocalizing the gastric lipase during the hydrolysis of NMFG. Two matrices of similar chemical composition but differing in structure were formulated: M1 containing NMFG and M2 obtained by homogenization and pasteurization of M1. The matrices were submitted to a semi-dynamic gastric in vitro digestion mimicking in vivo data. Rabbit Gastric lipase (RGL) was used as an analogue of HGL. The matrices disintegration and their hydrolysis kinetics were monitored. In parallel, RGL was immunolocalized using confocal or transmission electron microscopy after static in vitro gastric digestion of NMFG. The initial structure affected strongly the lipolysis kinetics, with rates approximately 40 times higher for M2 compared to M1. This was mainly explained by the highest specific surface developed by M2 (31 vs 1.8 m2/g of lipids). Quasi similar rates were obtained when normalized by the lipid specific surface (~ 0.2 µmol/min/m2) which confirmed the low sensitivity of RGL to the interfacial quality. The RGL immunolocalization backed up its high penetration capacity: discreet and specific locations of RGL insertion in the NMFG membrane were observed These differences of lipolysis kinetics may impact gastric emptying via ‘duodenal break’ and have physiological consequences. Optimizing infant formula specific surface to mirror the gastric behavior of human milk appears as a potential lever to improve infant nutrition.