Interactions between biological membranes and proteins are crucial in biological functions such as cell signaling, trafficking or in the control of membranes’ physical stability or functional properties. Over the last decades, the nanoscale resolution of Atomic Force Microscopy (AFM) has allowed direct observation of such interactions in near-physiological conditions[1]. Among biological membranes, the milk fat globule membrane (MFGM) is of special interest as the interface with the surrounding bulk of milk proteins during dairy processes or with enzymes and tissues of the gastro-intestinal tract during digestion. The MFGM is composed of polar lipids showing either zwitterionic, e.g. milk sphingomyelin (MSM) and phosphatidylcholine (PC), or anionic head groups, e.g. phosphatidylserine (PS) and phosphatidylinositol (PI). It exhibits lateral heterogeneity with microdomains of MSM in gel-phase or in liquid-ordered (Lo) phase in the presence of cholesterol, surrounded by the fluid matrix of glycerophospholipids, ie DOPC, in the liquid-disordered (Ld) phase[2,3]. In the present study, our goal was to determine how the phase and charge states of MFGM polar lipids drive interaction with the casein micelles, as the major milk protein 100-nm assembly, negatively charged at pH 6.7. For that, supported-hydrated lipid bilayers with various lipid compositions were prepared, including MFGM polar lipid extract, MSM, MSM/cholesterol, MSM/DOPC, DOPC/PS/PI. AFM topography imaging was performed before and after injection of casein micelles in the environment. Zeta-potential and Langmuir isotherms of the different polar lipids gave additional information necessary to interpret AFM observations. No interaction was observed between the casein micelles and the organized phases of MSM in gel-phase or MSM/cholesterol in Lo phase. But the casein micelles did adsorb onto the Ld phase of DOPC (figure 1), probably as a result of its larger inter-molecular distance. However, the presence of anionic polar lipids, PS and PI, in the Ld phase prevented such interaction by inducing electrostatic repulsion of the casein micelles. Thus, interaction of casein micelles with the MFGM is controlled by the phase state and the ionisation of its constitutive polar lipids. These results open perspectives for the design of emulsions and liposomes with variable capacity for protein adsorption, using milk polar lipids as food-grade ingredients.