Isolation of the industrially interesting milk fat globule membrane (MFGM) components from dairy streams is challenging, and a full exploitation of their benefits can be gained by better understanding of the behaviour of the fat globule membrane fraction during milk fat processing. In this study, microfiltration of the cream before churning and the comprehensive compositional analysis of the process streams revealed new insights on MFGM partitioning during the phase inversion in butter making. After removal of the smallest fat globules by microfiltration, a reduced phospholipid content was reflected in the buttermilk, but not in the butter serum. Regardless of the cream washing, buttermilk and butter serum lipids were different in PL-to-fat ratio, phospholipid composition, degree of unsaturation and melting behaviour. We suggest that partitioning of the MFGM fraction between butter and buttermilk is a direct result of the physico-chemical properties of the fat globules, partly related to the globule size.