Langmuir films and their analysis by tensiometry, ellipsometry and Langmuir–Blodgett transfer coupled to atomic force microscopy are suitable tools to study the interactions of various lipases with model membranes. The objective of our study was to characterize the adsorption and lipolysis behavior of different gastrointestinal enzymes onto biomimetic vegetal membranes. An heterogenous monolayer based on a mixture of unsaturated galactolipids (MGDG, DGDG), saturated phospholipids (DPPC), and phytosterols was used. Four enzymes were studied, (i) gastric lipase (GL), which has no lipolytic activity on polar lipids but whose adsorption may modulate the subsequent adsorption and activity of other lipases, and three pancreatic enzymes: (ii) pancreatic lipase 2 (PLRP2) presenting both galactolipase and phospholipase A1 activities, (iii) pancreatic triacylglycerol lipase and its cofactor, colipase (PTL/coPTL), and (iv) pancreatic secreted phospholipase A2 (sPLA2-IB). A strong surfactant property of GL and its preferential adsorption at both the expanded lipid phase and at the boundary phase were observed, in line with previous results obtained with biomimetic milk fat globule mem- brane. With PLRP2, changes in surface pressure indicated a lipolytic activity in addition to lipase adsorption, with a higher rate at surface pressure below 15 mN/m. A drastic decrease in film thickness was also observed by ellipsometry, suggesting the removal of some lipolysis products. Such variation was not observed upon PTL/coPTL adsorption and may be related to the absence of activity of PTL on polar lipids. Finally, the evolution of adsorption kinetic after injection of sPLA2-IB did not indicate a clear trend of lipolysis on the lipid film. However, a change in the morphology of condensed domains was observed by AFM, suggesting a phase separation of lipids induced by binding of sPLA2-IB. This study is a step forward in the understanding of interaction of gastrointestinal lipases with plant