The phospholipid and sphingolipid composition of milk is of considerable interest regarding their nutritional and functional properties. The objective of this article was to determine the lipid composition of the milk fat globule membrane (MFGM) of milk from cows fed a diet rich in polyunsaturated fatty acids. The experiments were performed with 2 groups of 6 cows feeding on (i) maize silage ad libitum (+ grassland hay, mixture of cereals, soyabean meal) or (ii) the maize silage-based diet supplemented with extruded linseed (bringing a lipid proportion of 5% of dry matter). The phospholipid and sphingolipid composition of the MFGM was determined using HPLC/ELSD. The fatty acid (FA) composition of total lipids and phospholipids was determined using GC. As expected, the linseed-supplemented diet decreased the saturated FA and increased the unsaturated FA content in milk fat. MFGM in milk from cows fed the diet rich in polyunsaturated FA resulted in (i) a higher amount of phospholipids (+ 18%), which was related to a smaller size of milk fat globules (ii) an increase of 30% (w/w) of the concentration in sphingomyelin, (iii) a higher content in stearic acid (1.7-fold), unsaturated FA (1.36-fold), and C18:1 trans FA: 7.2 +/- 0.5% (3.7-fold). The MFGM contained a higher concentration of unsaturated FA (C18:1, C18:2, and C18:3) and very long-chain FA (C22:0, C23:0, C24:0, EPA, DHA) compared with total lipids extracted from milk. The technological, sensorial, and nutritional consequences of these changes in the lipid composition of the MFGM induced by dietary manipulation remain to be elucidated.