Sphingomyelin-rich microdomains have been observed in the biological membrane surrounding milk fat globules (MFGM). The role played by cholesterol in these domains and in the physical properties and functions of the MFGM remains poorly understood. The objective of this work was therefore to investigate the phase state, topography, and mechanical properties of MFGM polar lipid bilayers as a function of cholesterol concentration, by combining X-ray diffraction, atomic force microscopy imaging, and force spectroscopy. At room temperature, i.e. below the phase transition temperature of the MFGM polar lipids, the bilayers showed the formation of sphingomyelin-rich domains in the solid ordered (so) phase that protruded about 1 nm above the liquid disordered (1(d)) phase. These so phase domains have a higher mechanical resistance to rupture than the l(d) phase (30 nN versus 15 nN). Addition of cholesterol in the MFGM polar lipid bilayers (i) induced the formation of liquid ordered (l(0)) phase for up to 27 mol % in the bilayers, (ii) decreased the height difference between the thicker ordered domains and the surrounding l(d) phase, (iii) promoted the formation of small sized domains, and (iv) decreased the mechanical resistance to rupture of the sphingomyelin-rich domains down to similar to 5 nN. The biological and functional relevance of the l(0) phase cholesterol/sphingomyelin-rich domains in the membrane surrounding fat globules in milk remains to be elucidated. This study brought new insight about the functional role of cholesterol in milk polar lipid ingredients, which can be used in the preparation of food emulsions, e.g. infant milk formulas.