The iTRAQ techniques were used to separate and identify milk fat globule membrane proteins(MFGMPs) from bovine and buffalo milk to compare the differences among composition, contents and functional properties of them. MFGMPs were separated and analyzed by Nano-HPLC tandem MS (MS/MS) analysis coupled with Q-Exactive. The results showed that 424 proteins were identified (FDR≤0.01%). The contents of 146 proteins were significantly different between MFGMPs bovine and buffalo in these identified proteins. Among these different proteins, there were 20 up regulated proteins and 25 down regulated proteins by two times (buffalo/bovine). The contents of sodium phosphate solute carrier protein and γ-glutamyl transpeptidase are significantly higher in buffalo MFGMPs than in bovine MFGMPs. The two proteins possess carriers and catalytic activities, and serve as the basic composition of biosynthesis and metabolism. The contents of xanthine oxidoreductase and butyrophilin are significantly higher in bovine MFGMPs than in buffalo MFGMPs. The two proteins, identified as binding protein, can inhibit the growth of tumor cell. The results showed that the significant differences of content existed in some functional protein between the MFGMPs of buffalo and bovine