Milk fat globule membrane conditions the reactivity and enzymatic susceptibility of milk lipids. The use of bovine membrane extracts to make infant formulas more biomimetic of human milk has been suggested recently. A comparison of the physico-chemical behavior of human and bovine milk membrane extracts and their interaction with gastric lipase is here undertaken using biophysical tools. Milk membrane extracts (70% of polar lipids) were obtained either pooling of mature human milk (n = 5) or bovine buttermilk. Human extract contained more anionic glycerophospholipids, less phosphatidylethanolamine and more unsaturated fatty acids (57% versus 46%) than bovine extract. Human extract presented a higher compressibility, with slower increase of surface pressure, than bovine extract. Micronic liquid condensed (LC) domains were evidenced in both extracts at 10 mN/m, but the evolution differs upon compression. Upon gastric lipase addition, an adsorption preference for liquid expanded phase (LE) was observed for both extracts. However, insertion was more homogeneous in terms of height level in human extract and impacted less its lipid lateral organization than in bovine extract. Both membrane extracts share close physico-chemical properties, however human membrane higher compressibility may favour gastric lipase insertion and higher interfacial reactivity in gastric conditions.