The proteins of the milk fat globule membrane (MFGM) have a number of functions, such as the regulation of milk fat secretion and metabolism, the uptake and transportation of fatty acids in the intestine and potential protection from bacterial or viral infection. While the proteome of the MFGM in bovine milk has been extensively characterised, our knowledge of these proteins in buffalo milk is limited. In this study, a proteomic approach was used to characterise the proteome of the buffalo MFGM. Multiple extraction techniques were employed to increase the coverage of proteins identified, while label free relative quantitative liquid chromatography tandem mass spectrometry was used for comparison between the buffalo and bovine MFGM proteome. A total of 220 buffalo MFGM proteins and 234 bovine MFGM proteins were identified after being filtered from the initial dataset of 757 and 680 proteins respectively. Significantly greater amounts of xanthine oxidoreductase, platelet glycoprotein 4, heat shock cognate and calcineurin B homologous protein were identified per mass of buffalo MFGM protein extracted. The higher expression of xanthine oxidase in the MFGM of buffalo milk was confirmed by Western blot analysis and a heterogeneous distribution of this protein observed in situ on the surface of the MFGM. The high concentration of fat in buffalo milk, together with the differences in the MFGM proteome indicate differences in the nutritional profile, biological function, and potential ease of processing of buffalo and bovine milk products.