Fat is present in milk as droplets of triacylglycerols surrounded by a complex membrane derived from the mammary epithelial cell called the milk fat globule membrane (MFGM). In-depth proteomic studies have been published for bovine MFGM proteins. However, to date, only sparse studies exist on MFGM proteins from non-cow milk. We have therefore characterized MFGM proteins in goat, sheep, horse and camel milk and we have highlighted prominent differences across species, especially for lactadherin, a major protein of the milk fat globule membrane. Indeed, lactadherin from goat and sheep milk appears as a single polypeptide chain in 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), whereas two and four polypeptide chains are respectively identified for lactadherin from camel and horse milk. Implications of the findings, especially with regard to bioactive properties of milk are discussed.